JP6948660B2 - Method of introducing foreign substances into cells and materials used for the method - Google Patents

Description

The present invention relates to a method for introducing a foreign substance into a cell and a material used for the method. More specifically, the present invention relates to a method for introducing a foreign substance into a target cell by utilizing the uptake of an exosome (exosome) by macropinocytosis, and a material used for the method.
This application claims priority based on Japanese Patent Application No. 2014-230472 filed on November 13, 2014, and the entire contents of the Japanese application are incorporated herein by reference. ing.

In the field of pharmacy, the development of technology for controlling the distribution of drugs in the body spatially, temporally, and quantitatively is one of the very important themes. By appropriately controlling the drug distribution in the body, for example, it is possible to enhance the pharmacological effect, reduce side effects, reduce the number of administrations, reduce costs, and the like. Such a technique for controlling the distribution of drugs in the body spatially, temporally, and quantitatively is generally called a drug delivery system (DDS), and is being actively researched and developed.

In recent years, a technique related to DDS at the cellular level, typically a technique for efficiently introducing a foreign substance into a target cell, has attracted attention. By applying such a technique, for example, it becomes possible to efficiently supply (act) a target drug to the causative cells of a disease. In particular, if a drug having strong side effects on normal cells (for example, an anticancer drug) can be efficiently acted on target cells (for example, cancer cells), it is possible to realize a high effect while reducing side effects. can.
In addition, such technology related to DDS at the cellular level has attracted a great deal of attention in the field of regenerative medicine. For example, by applying such a technique, a reprogramming factor, a differentiation-inducing factor, or the like can be efficiently introduced into a target cell. In addition, when a transplant material for regenerative medicine (for example, a stem cell used for regenerative medicine, or a cell or tissue induced to differentiate from the stem cell) is transplanted into a living body, before the transplanted material is engrafted in the living body. It is also possible to apply necessary factors and the like to efficiently supply the transplant material.
Patent Documents 1 to 3 describe a technique for introducing a desired substance (typically a drug) into cells using various carrier substances as a technique relating to DDS at the cell level.

Japanese Unexamined Patent Publication No. 2013-121986 Japanese Unexamined Patent Publication No. 2013-06392 Special Table 2003-517886

Chemistry & Biology, Vol. 19, 2012, pp. 1437-1446 The Journal of Biological Chemistry, Volume 289, 2014, pp. 22258-22267 Journal of Cell Science, Vol. 94, 1989, pp.135-142

By the way, for example, in the conventional DDS described in Patent Documents 1 to 3 and the like, an in vitro compound is generally used as a carrier substance for introducing a foreign substance into a cell. When such an in vitro compound is used, a sufficient effect may not be exhibited due to cytotoxicity derived from such a carrier, an immune reaction against the carrier, and the like.
For this reason, attempts have been made to establish a technique for introducing a foreign substance into a cell using a substance derived from a living body.

In view of such circumstances, the present invention has been created for the purpose of providing a novel material used for introducing a foreign substance into a cell. Specifically, the purpose is to establish a technique for introducing a foreign substance into a living body by using a substance derived from the living body. Another object is to provide a method for introducing a foreign substance into a cell of interest using such a material.

In order to achieve the above object, the present inventors have focused on exosomes that are widely distributed in the living body. Exosomes are vesicles made of lipid bilayer membranes that are present in the body (typically in body fluids). The present inventors have enthusiastically promoted research on the mechanism by which exosomes are taken up into cells, and by inducing macropinocytosis in the target cells, foreign substances contained in the exosomes are suitable for the target cells. Found that it could be introduced in. Furthermore, the present inventors have newly found that by supplying a target cell with an exosome containing a substance that induces macropinocytosis, the uptake of the exosome in the target cell is promoted. Based on these findings, the present inventors have completed the present invention.

Here, Non-Patent Document 1 describes a phenomenon in which macropinocytosis is activated by supplying a substance that stimulates a specific receptor to a cell, and Non-Patent Document 2 describes a macro. The phenomenon that exosomes are taken up into cells by pinocytosis is described. However, by inducing macropinocytosis, foreign substances contained in exosomes can be suitably introduced into target cells, and exosomes containing substances that induce macropinocytosis can be supplied to cells. There is no disclosure or suggestion that exosome uptake in the cells is promoted.

According to the present invention, in order to achieve the above object, a foreign substance is introduced into the target cell when it is supplied to the target cell (typically when it is added to the medium in which the cell is cultured). Exosomes having the ability (hereinafter, such exosomes are also referred to as “exosomes for introducing foreign substances”) are provided.
That is, the exosome disclosed here (exosome for introducing a foreign substance) is an exosome used for introducing a foreign substance into a target cell from the outside of the cell into the cell.
With one or more foreign substances
Substances that induce macropinocytosis in target cells and
It is an exosome that contains.

The exosome for introducing a foreign substance disclosed herein is characterized by containing a substance that induces macropinocytosis and the foreign substance in the exosome. This makes it possible to induce (activate) macropinocytosis in the target cell and promote the uptake of the exosome by the target cell. Therefore, the foreign substance contained in the exosome for introducing the foreign substance can be efficiently introduced into the target cell.
Here, the exosome is a substance derived from a living body. Therefore, the cytotoxicity of exosomes and the immune response to exosomes can be reduced as compared with substances derived from in vitro compounds. For this reason, exosomes are preferred as carriers for introducing foreign substances.
Further, since the exosome for introducing a foreign substance disclosed here contains a target foreign substance and a substance that induces macropinocytosis in the exosome, the substance that induces macropinocytosis and the foreign substance are contained in the exosome. It is not necessary to perform an operation of supplying the contained exosomes to the target cells, or an operation of mixing them. Therefore, the target foreign substance can be introduced into the target cell by a simple operation of supplying the exosome for introducing the foreign substance to the target cell.
Exosomes are vesicles that can stably maintain their structure in cell culture medium. In addition, it is difficult to be decomposed even in a living body (for example, in blood) and can exist stably. Therefore, by including a substance that induces macropinocytosis and a foreign substance in the exosome, it is possible to reduce the decomposition (metabolism, inactivation) of these substances. For example, drug metabolism in the liver, excretion as excrement, elimination by immune cells, etc. can be suitably reduced (avoided). Therefore, according to the exosome for introducing a foreign substance disclosed here, a substance that induces macropinocytosis and a foreign substance can be stably supplied to cells (particularly cells in a living body).
Therefore, the exosome for introducing a foreign substance disclosed here is preferable as a component of a composition used for introducing a foreign substance into a target cell. It can also be used as a method for introducing a foreign substance into a target cell.

In a preferred embodiment of the exosomes disclosed herein (exosomes for introducing foreign substances), the substance that induces macropinocytosis in the target cells stimulates the epidermal growth factor receptor (EGFR). A substance that stimulates (activates) CXC Chemokine receptor 4: CXCR4, or CD184 or Fusin.
Epidermal growth factor receptor and CXC chemokine receptor 4 are typical examples of receptors that can induce macropinocytosis by stimulating the receptor. Therefore, the substance that stimulates (activates) the epidermal growth factor receptor and the substance that stimulates (activates) the CXC chemokine receptor 4 are suitable as substances that induce macropinocytosis.
Among them, EGF or its analog, or SDF (stromal cell-derived factor) or an exosome containing the analog as a substance that induces macropinocytosis in the target cell is disclosed here. This is a preferred embodiment of the exosome to be used (exosome for introducing a foreign substance). EGF or its analog is a substance that preferably stimulates the epidermal growth factor receptor, and SDF or its analog is a substance that preferably stimulates the CXC chemokine receptor 4.
In general, chemokines have four cysteine residues (C) conserved in the molecule, four depending on the difference in the sequence relationship between the two N-terminal cysteine residues and other amino acid residues. It is classified into subfamilies (C, CC, CXC, CX3C). Of these, the CXC chemokine has an amino acid sequence (“CX—C” sequence) in which one arbitrary amino acid residue (X) is present between the two cysteine residues (C) on the N-terminal side. A chemokine that belongs to a subfamily.

In a preferred embodiment of the exosome (exosome for introducing a foreign substance) disclosed herein, the foreign substance is a substance having pharmacological activity. With such a configuration, it is possible to efficiently supply a substance having pharmacological activity to the target cells. As a result, the pharmacological effect of the substance having pharmacological activity can be exhibited at a high level.

In a preferred embodiment of the exosome (exosome for introducing a foreign substance) disclosed herein, the target cell is a human tumor cell, and the foreign substance is a compound having antitumor activity. Many compounds having antitumor activity typically have strong side effects on normal cells. According to this aspect, since the compound having antitumor activity can be efficiently supplied to human tumor cells, the antitumor activity against tumor cells is reduced while the side effects of the compound having antitumor activity on normal cells are reduced. Can be demonstrated at a high level.

In addition, as another aspect of the present invention, a composition used for introducing a foreign substance into a target cell from the outside of the cell into the cell (hereinafter, such a composition is referred to as "a composition for introducing a foreign substance". (Also called)
With one or more pharmaceutically acceptable carriers
Substances that induce macropinocytosis in target cells and
Exosomes containing one or more foreign substances and
To provide a composition (pharmaceutical composition) comprising.
Since the composition having such a constitution (composition for introducing a foreign substance) contains a substance that induces macropinocytosis, it is possible to suitably promote the uptake of exosomes into a target cell. As a result, the foreign substance contained in the exosome can be efficiently introduced into the target cell.

Further, as another aspect, the present invention is a composition (composition for introducing a foreign substance) used for introducing a foreign substance into a target cell from the outside of the cell into the cell.
With one or more pharmaceutically acceptable carriers
Exosomes containing both substances that induce macropinocytosis in target cells and one or more foreign substances, and
To provide a composition (pharmaceutical composition) comprising.
Since the composition having such a constitution (composition for introducing a foreign substance) contains a substance that induces macropinocytosis in the exosome, the uptake of the exosome can be suitably promoted to the target cell. As a result, the foreign substance contained in the exosome can be efficiently introduced into the target cell. Furthermore, since a substance that induces macropinocytosis with respect to the target cell is included in the exosome, the substance that induces macropinocytosis can be stably supplied to the target cell.

Further, as another aspect, the present invention is a method for introducing a foreign substance into a target cell in vitro.
Prepare a cell culture containing the target cells, and include in the cell culture a substance that induces macropinocytosis with respect to the target cells and one or more foreign substances. Supplying exosomes,
Provide a method including.
According to the method for introducing a foreign substance, the foreign substance is introduced into a target cell with high efficiency by a simple method of supplying exosomes into a culture containing the target cells (typically in a medium for culturing the cells). can do. Further, by using an exosome, which is a substance derived from a living body, as a carrier for introducing a foreign substance into a cell, cytotoxicity due to the carrier can be reduced.

FIG. 1 shows a confocal laser scanning microscope for uptake of exosomes by the cells when the exosomes (sample 1) according to one test example were supplied to the target cells (A431 cells) and cultured in the presence or absence of EGF. It is a micrograph (image) examined using a microscope. The photograph (image) shown on the left side (row of CD63-GFP exosome) is a fluorescence micrograph (FL image) in which the localization of exosomes was examined by fluorescence observation. The photograph (image) shown in the right column (Merge column) is an image in which the FL image shown in the left column and the nuclear-stained image by Hoechst33342 are superimposed (merged). The scales in the photographs are all 20 μm. FIG. 2 shows the uptake of exosomes by the cells when the exosomes (sample 1) according to one test example were supplied to the target cells (A431 cells) and cultured under different medium conditions (presence or absence of FBS and EGF concentration). It is a graph which shows the result which examined by the analysis using the flow cytometer. In such a graph, the average GFP fluorescence intensity per cell in each test group is relative to the average GFP fluorescence intensity per cell in the control group (test group cultured under medium conditions without addition of FBS and EGF). Shown by ratio. The average value when the same test is repeated three times independently is shown by a bar graph, and the standard deviation (± SD) is shown by an error bar. FIG. 3 shows a confocal laser scanning microscope for uptake of exosomes and dextran by the cells when the exosomes (sample 1) and dextran according to one test example were supplied to the target cells (A431 cells) and cultured in the presence of EGF. It is a micrograph (image) examined using a microscope. The photograph (image) shown in the upper left (Exosome) is a fluorescence micrograph in which the localization of exosomes is examined by fluorescence observation. The photograph (image) shown in the lower left (Dextran) is a fluorescence micrograph in which the localization of dextran was examined by fluorescence observation. The photograph (image) shown on the right (Merge) is an image in which the image (Exosome) shown on the upper left and the image (Dextran) shown on the lower left are superimposed (merged). The arrows in the photographs (images) point to typical examples of regions where exosomes and dextran taken up into cells are co-localized. The scales in the photographs are all 10 μm. In FIG. 4, 5- (N-ethyl-N-isopropyl) amiloride (hereinafter, also referred to as “EIPA”), which is an inhibitor of macropinocytosis, is added to the medium and cultured, and then one test example is applied. It is a graph which shows the result of having examined the uptake of an exosome by the cell when the exosome (sample 1) was supplied to the target cell (A431 cell) and cultured by the analysis using the flow cytometer. Such a graph shows the average GFP fluorescence intensity per cell in the EIPA-treated group as a relative ratio to the average GFP fluorescence intensity per cell in the control group (EIPA untreated group). The average value when the same test is repeated three times independently is shown by a bar graph, and the standard deviation (± SD) is shown by an error bar. FIG. 5 shows the cell viability (%) of the cells when the exosome (Sample 1) according to one test example was supplied to the target cells (A431 cells) and cultured at different EGF concentrations in the medium. It is a graph which shows the result. Such a graph shows the cell viability (%) in each test group as a relative ratio to the cell viability (%) in the control group (the test group cultured under the culture conditions to which neither exosome nor EGF was added). The average value when the same test is repeated three times independently is shown by a bar graph, and the standard deviation (± SD) is shown by an error bar. FIG. 6 shows the cell viability (%) of the cells when the exosome (sample 2) or saporin according to one test example was supplied to the target cells (A431 cells) and cultured in the presence or absence of EGF. It is a graph which shows the result of having investigated. Such a graph shows the relative ratio of the cell viability (%) in each test group to the cell viability (%) in the control group (test group cultured under culture conditions without addition of exosomes, saporins, and EGF). show. The average value when the same test is repeated three times independently is shown by a bar graph, and the standard deviation (± SD) is shown by an error bar. FIG. 7 shows the uptake of exosomes by the cells when the exosomes (samples 1, 3, 5) according to one test example were supplied to the target cells (A431 cells) and cultured by analysis using a flow cytometer. It is a graph which shows the result of the investigation. Such a graph shows the average GFP fluorescence intensity per cell in each test group as a relative ratio to the average GFP fluorescence intensity per cell in the sample 1 addition group. The average value when the same test is repeated three times independently is shown by a bar graph, and the standard deviation (± SD) is shown by an error bar. FIG. 8 is a graph showing the results of examining the cell viability (%) of the exosomes (samples 2 or 4) according to one test example when the target cells (A431 cells) were supplied and cultured. .. In such a graph, the horizontal axis shows the exosome concentration in the medium, and the vertical axis shows the cell viability (%) in each test group, and the control group (test group cultured under culture conditions without exosome addition and EGF addition). It is shown as a relative ratio with the cell viability (%) in. The mean values when the same test is repeated three times independently are plotted and shown, and the standard deviation (± SD) is shown by error bars. FIG. 9 is a photomicrograph (image) of the morphology of the target cells when the exosomes (samples 2 or 4) according to one test example were supplied to the target cells (A431 cells) and cultured using an optical microscope. show. The concentration (μg / mL) in the figure indicates the exosome concentration in the medium. FIG. 10 shows a flow site regarding the uptake of exosomes by the cells when the exosomes (sample 1) according to one test example were supplied to the target cells (A431 cells) and cultured for 96 hours at different EGF concentrations in the medium. It is a graph which shows the result which examined by the analysis using the meter. Such a graph shows the average GFP fluorescence intensity per cell in each test group as a relative ratio to the average GFP fluorescence intensity per cell in the control group (EGF-free group). The average value when the same test is repeated three times independently is shown by a bar graph, and the standard deviation (± SD) is shown by an error bar. FIG. 11 shows the uptake of exosomes by the cells when the exosomes (sample 1) according to one test example were supplied to the target cells (A431 cells) and cultured for 96 hours in the presence of EGF or in the absence of EGF. It is a micrograph (image) examined using a confocal laser scanning microscope. The photograph (image) shown in the left column (column of CD63-GFP exosome) is a fluorescence micrograph (FL image) in which the localization of exosomes is examined by fluorescence observation. The photograph (image) shown in the central row (row of Hoechst33342) is a nuclear-stained image by Hoechst33342. The photograph (image) shown in the right column (Merge column) is an image in which the FL image shown in the left column and the nuclear stained image shown in the center column are superimposed (merged). The scales in the photographs are all 20 μm. FIG. 12 is a graph showing the results of examining the uptake of exosomes (sample 1) according to one test example by MIA PaCa-2 cells as target cells by analysis using a flow cytometer. be. Such a graph shows the average GFP fluorescence intensity per cell in each test group as a relative ratio to the average GFP fluorescence intensity per cell in the control group (EGF-free group). The average value when the same test is repeated three times independently is shown by a bar graph, and the standard deviation (± SD) is shown by an error bar. FIG. 13 is a photomicrograph (image) of the uptake of exosomes (sample 1) and dextran according to one test example by the cells when MIA PaCa-2 cells were used as the target cells, examined using a confocal laser scanning microscope. ). The photograph (image) shown on the far left (Exosome) is a fluorescence micrograph (image) in which the localization of exosomes is examined by fluorescence observation. The photograph (image) shown in the second from the left (Dextran) is a fluorescence photograph (image) in which the localization of dextran was examined by fluorescence observation. The photograph (image) shown in the second from the right (Hoechst33342) is a nuclear-stained image by Hoechst33342. The photographs (images) shown in the right column (Merge column) are the leftmost image (Exosome), the second image from the left (Dextran), and the second nuclear stain from the right. It is an image that is superimposed (merged) with the image. The scales in the photographs are all 10 μm. FIG. 14 is a graph showing the results of examining the uptake of exosomes (sample 1) according to one test example by BxPC-3 cells as target cells by analysis using a flow cytometer. .. Such a graph shows the average GFP fluorescence intensity per cell in each test group as a relative ratio to the average GFP fluorescence intensity per cell in the control group (EGF-free group). The average value when the same test is repeated three times independently is shown by a bar graph, and the standard deviation (± SD) is shown by an error bar. FIG. 15 shows a flow cytometer for uptake of exosomes by the cells when the exosomes (sample 1) according to one test example were supplied to the target cells (HeLa cells) and cultured at different SDF concentrations in the medium. It is a graph which shows the result examined by the analysis used. Such a graph shows the average GFP fluorescence intensity per cell in each test group as a relative ratio to the average GFP fluorescence intensity per cell in the control group (SDF-free group). The average value when the same test is repeated three times independently is shown by a bar graph, and the standard deviation (± SD) is shown by an error bar.

Hereinafter, preferred embodiments of the present invention will be described. Matters other than those specifically mentioned herein (eg, the composition of the exosomes for introducing foreign substances and the compositions for introducing foreign substances disclosed herein) and necessary for the practice of the present invention (eg, desired ones). General matters such as methods of synthesizing and obtaining foreign substances, cell culture techniques, preparation of pharmaceutical compositions) include cell engineering, physiology, medicine, pharmacy, organic chemistry, biochemistry, genetic engineering, protein engineering, molecules. It can be grasped as a design matter of a person skilled in the art based on the prior art in the fields of biology, genetics, and the like. The present invention can be carried out based on the contents disclosed in the present specification and common general technical knowledge in the art.
In addition, all the contents of all the documents cited herein are incorporated herein by reference.

As used herein, the term "exosome" means a vesicle made of a lipid bilayer that is secreted extracellularly from various eukaryotic cells. Typically, it is a vesicle having a diameter of about 50 nm to 100 nm. Exosomes are clearly distinguished from liposomes (vesicles artificially created to imitate cell membranes) in that they are vesicles secreted by cells (ie, substances of biological origin).
The cells that secrete exosomes are not particularly limited, and for example, various cultured cells (for example, cell lines, primary cultures, etc.) and various cells existing in vivo (for example, epithelial cells, fibroblasts, muscle cells, etc.) Immune system cells, hematopoietic cells, etc.) can secrete exosomes. Not only normal cells but also tumor cells, pathogen-infected cells and the like can secrete exosomes. Such exosomes are present in the medium in which the cells are cultured and in any body fluid in vivo. Examples of the body fluid include blood (typically serum and plasma), saliva, urine, pleural effusion, bone marrow, and amniotic fluid. Exosomes can be present stably in such body fluids and in culture (without degradation of inclusions in body fluids).

As used herein, "macropinocytosis" is a type of endocytosis, and is an extracellular substance (including extracellular fluid) in which the cell membrane protrudes due to the polymerization of actin (actin filament) around the cell membrane. Is taken up into cells. Typically, membrane ruffling is observed.
Here, the above-mentioned macropinocytosis and endocytosis in which an extracellular substance is taken into a cell in the form of a cell membrane depressed into the cell are clearly distinguished in the following points. The cell membrane depressed endocytosis typically progresses in a clathrin protein-dependent manner on the cell membrane surface. For this reason, it is also called clathrin-dependent endocytosis. On the other hand, macropinocytosis is clathrin protein-independent. In addition, the endosomes formed by clathrin-dependent endocytosis have a diameter of about 100 nm, whereas the endosomes formed by macropinocytosis (also called macropinosomes) have a diameter of about 200 nm or more. It is as large as 5000 nm (0.2 μm to 5 μm).

As used herein, the term "foreign substance" means a foreign substance as seen from the target cell to which the present invention is applied. That is, if the target cell is a cultured cell maintained in an in vitro culture system, it cannot be distinguished by whether or not the target cultured cell is a foreign substance in the isolated organism. For example, any substance that is introduced into a target cultured cell from the outside of the cell can be a foreign substance. For example, even a substance isolated from the same cell line (cell line) as the target cell can be a foreign substance to the target cell in that the substance is introduced into the cell from the outside of the cell. Further, if the target cell is a cell existing in a living body, it does not matter whether or not the target cell is a foreign substance in the living body (individual) in which the cell exists. For example, it was isolated not only from a substance artificially synthesized outside the living body in which the target cell exists, a substance derived from a living body other than the living body in which the target cell exists, but also from a living body (same individual) in which the target cell exists. The substance is also a typical example of the above-mentioned foreign substance.

The exosomes disclosed herein are exosomes first discovered by the present inventor that they can be used to introduce foreign substances into target cells. As described above, the exosomes disclosed herein are one or more foreign substances and a substance that induces macropinocytosis in a target cell (hereinafter, also referred to as "macropinocytosis inducer"). It is an exosome containing and (an exosome for introducing a foreign substance). That is, the exosomes for introducing foreign substances disclosed herein are typically exosome particles as carriers, foreign substances contained in the exosome particles, and macropinocytosis contained in the exosome particles. Includes inducers. Hereinafter, each substance constituting the exosome for introducing a foreign substance will be described below.

<Exosomes>
As the exosomes (that is, exosome particles as carriers) used in the exosomes for introducing foreign substances disclosed here, exosomes secreted from various cells can be recovered and used. For example, biofluids such as blood (typically serum), urine, saliva, lymph, gastrointestinal fluid, lung lavage fluid, cerebrospinal fluid, inflammatory discharge fluid, amniotic fluid, and breast milk contain exosomes of biological origin. .. Therefore, such a biological fluid can be collected, and exosomes in the biological fluid can be recovered and used. Alternatively, since exosomes released (secreted) from the cultured cells are also present in the culture medium in which the cells are cultured, the exosomes recovered from the cell culture medium can be used without particular limitation.

The exosomes recovered from the biological fluid and the exosomes recovered from the cell culture medium can be used without distinction from the viewpoint of introducing the foreign substance in the present invention into the target cells. It can be appropriately selected and used. From the viewpoint of efficiently recovering (preparing) a required amount of exosomes, it is preferable to use exosomes recovered from the cell culture medium.

Further, depending on the type of cell secreting the exosome, the properties of the exosome, for example, the profile of the lipid bilayer membrane constituting the exosome (for example, the peptide (including protein), lipid, sugar contained in the lipid bilayer membrane). The composition of the chain, etc.) and the composition of substances originally contained in the exosome (for example, peptides (including proteins), RNA (mRNA, microRNA, etc.), soluble components contained in the cytoplasm, etc.) may differ. be. Therefore, when using exosomes having specific properties, it is preferable to use exosomes recovered from the culture medium in which specific cells are cultured. As a result, exosomes having similar properties can be obtained in a highly pure state.

Alternatively, a specific molecule may be imparted to the surface of the membrane constituting the exosome by a genetic engineering technique or the like. For example, by forcibly expressing a specific molecule (for example, peptide, protein, etc.) on the cell membrane surface of an exosome-secreting cell (exosome-producing cell), the target molecule can be imparted to the surface of the membrane constituting the exosome. .. By adopting a molecule that acts on a molecule that is highly expressed in the target cell (typically, it binds to a receptor that is highly expressed in the target cell) as such a specific molecule, a foreign substance is highly efficiently applied to the target cell. Can be introduced. That is, it is preferable because the specificity for the target cell is improved.

As a method for recovering exosomes from the above-mentioned biological fluid and cell culture solution, a conventionally known method can be adopted. For example, exosomes can be isolated by ultracentrifugation (Thery C., Curr. Protoc. Cell Biol. (2006) Chapter 3: Unit 3.22.). Alternatively, exosomes may be isolated using commercially available reagents (kits) such as Total esosome isolation (from cel culture media) (manufactured by Life Technologies (Invitrogen)). The above-mentioned biological fluid and cell culture fluid can also be obtained by a method using an antibody against an exosome-specific marker protein (for example, CD9, CD63, CD81, etc.), a method using an appropriate filter or column (filter method, column method, etc.), etc. Exosomes can be recovered from the inside. Examples thereof include an immunoprecipitation method, a FACS method, an ultrafiltration method, a gel filtration method, a filter method, an HPLC method, a method of precipitating exosomes adsorbed on a polymer or beads, or a method of separating them using a column.

The recovery of exosomes by the above-mentioned method is observed using a transmission electron microscope (typically negative staining), or a method such as Western blotting, ELISA, FACS (typically immunological). Method) includes a method of detecting an exosome marker protein (for example, CD9, CD63, CD81, etc.). Alternatively, RNA derived from exosomes may be measured.

Here, in the present specification, the amount of exosomes (g) recovered by the above-mentioned method is shown in terms of the total amount of protein of the recovered exosomes. That is, the amount of recovered exosomes (g) can be calculated by measuring the total amount of protein in the recovered exosome dispersion. Specifically, when the recovered exosome dispersion contains 1 μg / mL of total protein, it is treated as if the exosome dispersion contains 1 μg / mL of exosome. do.

<Macropinocytosis inducer>
The macropinocytosis-inducing substance used in the exosome for introducing a foreign substance disclosed here is not particularly limited as long as it is a substance capable of inducing macropinocytosis in a target cell.
For example, it may be a substance that stimulates (activates) the Epidermal Growth Factor Receptor (EGFR) or a substance that stimulates (activates) CXC Chemokine Receptor 4: CXCR4. Alternatively, a substance that stimulates (activates) Platelet Derived Growth Factor Receptor (PDGFR), Fibroblast Growth Factor Receptor (FGFR) and / or Syndecan-4 (Syndecan). -4) can be a substance that stimulates (activates). EGFR, CXCR4 and PDGFR are all receptors that can induce macropinocytosis by stimulating (activating) the receptor. In addition, FGFR and Syndecan-4 are receptors that can induce macropinocytosis by stimulating (activating) these receptors (or complexes of these receptors).
Alternatively, a substance known to be capable of inducing macropinocytosis without requiring stimulation of a specific receptor can also be adopted as the macropinocytosis inducing substance. For example, it can be a substance that activates intracellular PKC (Protein Kinase C) without requiring stimulation of a specific receptor. Non-Patent Document 3 describes that macropinocytosis can be induced by activating the intracellular PKC of a target cell.

Here, as the substance that stimulates EGFR, a substance conventionally known to activate EGFR can be used without particular limitation. Typically, a substance known as a ligand for EGFR can be used. Here, as the ligand of EGFR, epidermal growth factor (EGF), α-type transforming growth factor (α, TGF-α), heparin binding epidermal growth factor-like growth factor (heparin binding). -Epidermal growth factor-like growth factor (HB-EGF), amphiregulin (AREG, AR), betacellulin (BTC), epiregulin (EREG), epigen (Epigen, EPG) and the like.
Such a ligand of EGFR and an analog thereof are substances that can be suitably used as a macropinocytosis inducer. Alternatively, agonists of these EGFR ligands and their analogs can also be suitably used as macropinocytosis inducers.

Among them, EGF and its analog, or an EGF agonist and its analog are excellent in the ability to induce macropinocytosis by stimulating EGFR, and thus can be suitably used as a macropinocytosis inducer. In particular, EGF and its analogs are suitable for practicing the present invention.

Further, as the substance that stimulates CXCR4, a substance conventionally known to activate CXCR4 can be used without particular limitation. Typically, a substance known as a ligand for CXCR4 can be used. Here, as an example of a substance that stimulates CXCR4 (typically, a ligand of CXCR4), Stromal cell-derived factor-1 (SDF-1) can be mentioned.
Such a ligand of CXCR4 (typically SDF-1) and an analog thereof are substances that can be suitably used as a macropinocytosis inducer. Alternatively, such CXCR4 ligand (typically SDF-1) agonists and analogs thereof may also be suitably used as macropinocytosis inducers. In particular, SDF-1 and its analogs are suitable for practicing the present invention.

Further, as the substance that stimulates PDGFR, a substance conventionally known to activate PDGFR can be used without particular limitation. Typically, a substance known as a ligand for PDGFR may be used. Here, examples of a substance that stimulates PDGFR (typically a ligand of PDGFR) include Platelet Derived Growth Factor (PDGF).
Such PDGFR ligands (typically PDGF) and their analogs are substances that can be suitably used as macropinocytosis inducers. Alternatively, agonists of such PDGFR ligands (typically PDGF) and analogs thereof may also be suitably used as macropinocytosis inducers. In particular, PDGF and its analogs are suitable for practicing the present invention.

Further, as the substance that stimulates FGFR and / or Syndecan-4, a substance conventionally known to activate at least any of FGFR and Syndecan-4 can be used without particular limitation. Typically, substances known as ligands for FGFR and Syndecan-4 can be used. Here, examples of substances that stimulate FGFR and Syndecan-4 (typically, ligands for FGFR) include Fibroblast Growth Factor (FGF).
Such FGFR and Syndecan-4 ligands (typically FGF) and their analogs are substances that can be suitably used as macropinocytosis inducers. Alternatively, such FGFR and Syndecan-4 ligand (typically FGF) agonists and analogs thereof may also be suitably used as macropinocytosis inducers. In particular, FGF and its analogs are suitable for practicing the present invention.

Further, as the substance that activates the intracellular PKC without requiring stimulation of the specific receptor, a substance conventionally known to activate the intracellular PKC is used without particular limitation. can do. Examples of the substance that activates the intracellular PKC include phorbol ester (specifically, Phorbol 12-Myristate 13-Acetate (PMA)). The phorbol ester (typically PMA) is a substance known to induce macropinocytosis by activating intracellular PKC (Non-Patent Document 3).

The macropinocytosis-inducing substance can be appropriately selected depending on the target cell. For example, if the target cell is a cell expressing EGFR (typically a cell highly expressing EGFR), a substance that stimulates EGFR can be preferably adopted as a macropinocytosis inducer. Further, if the target cell is a cell expressing CXCR4 (typically a cell highly expressing CXCR4), a substance that stimulates CXCR4 can be preferably adopted as a macropinocytosis inducer. As in the case of EGFR and CXCR4, if the target cell is a cell expressing PDGFR (typically a cell highly expressing PDGFR), a substance that stimulates PDGFR is preferably adopted as a macropinocytosis inducer. And if the target cell is a cell expressing FGFR and / or Syndecan-4 (typically a cell expressing both FGFR and Syndecan-4), macropinocytosis induction As a substance, a substance that stimulates FGFR and / or Syndecan-4 can be preferably adopted.

The substance that induces macropinocytosis disclosed herein may be in the form of a salt as long as the ability to induce macropinocytosis with respect to the target cells is not impaired. For example, an acid addition salt that can be obtained by an addition reaction of an inorganic acid or an organic acid that is usually used according to a conventional method can be used. Alternatively, other salts (for example, metal salts) may be used as long as they have the ability to induce macropinocytosis in the target cells. Thus, the "substances that induce macropinocytosis" as described herein and in the claims include those in such salt form.

The substance that induces macropinocytosis (typically, the substance that stimulates EGFR, the substance that stimulates CXCR4, the substance that stimulates PDGFR, the substance that stimulates FGFR and Syndecan-4, and the substance that stimulates Syndecan-4, and the above. As the substance that activates PKC in the cell), a substance obtained by purchasing a commercially available product can be used. Alternatively, it may be produced by a known method. For example, it can be a substance artificially synthesized by chemical synthesis or biosynthesis (for example, production based on genetic engineering). Since the method for obtaining the substance that induces macropinocytosis does not characterize the present invention, detailed description thereof will be omitted.

<Foreign substance>
The exosome for introducing a foreign substance disclosed here contains one or more (for example, three or more) desired foreign substances in the exosome. The foreign substance is not particularly limited as long as it is a substance that is desired to be introduced into the target cell. A substance having some physiological activity (typically pharmacological activity) is preferable because it has high utility value in the medical industry. From the viewpoint of using an exosome for introducing a foreign substance for a therapeutic purpose for a specific disease, a substance exhibiting a therapeutic effect on the disease is preferable as the foreign substance in the present invention. For example, it can be a drug or a substance used as an active ingredient of the drug. The drug or its active substance is not particularly limited as long as it is a substance approved for use as a drug, and for example, a chemically synthesized drug (typically a small molecule drug) and a biotechnology applied drug (so-called biopharmaceutical). ), Crude drug (for example, Chinese herbal medicine) and the like.

The physiological activity (typically pharmacological activity) of the foreign substance may be a property that acts on a specific physiological regulatory function on a target cell. Alternatively, it may have the property of suppressing the growth of a specific pathogen. For example, antitumor activity, predetermined differentiation-inducing activity, differentiation-suppressing activity, dedifferentiation-inducing activity, cell proliferation (division) promoting activity, cell proliferation (dividing) suppressing activity, anti-angiogenic effect, angiogenesis-promoting effect, immunosuppressive effect. , Immunostimulatory action, antiallergic action, anti-inflammatory action, hematopoietic promoting action, psychotropic action, antidepressant action, hormone-like action, antihormonal action, promoting action for specific enzymatic reaction, inhibitory action for specific enzymatic reaction, specific It may have a promoting action on signal transduction, an inhibitory action on specific signal transduction, and the like. Alternatively, antibacterial action, antiviral action, antifungal action, antiparasitic action and the like can be mentioned. Alternatively, it may have a diabetes improving action, a hypertension improving action, a hyperlipidemia improving action, an antithrombotic action, an analgesic action, a diuretic action and the like.

Therefore, the foreign substances include antitumor substances (anticancer substances), differentiation-inducing substances, differentiation-inhibiting substances, dedifferentiation-inducing substances, cell growth (division) -promoting substances, cell growth (division) -suppressing substances, and antiangiogenic substances. Substances, angiogenesis-promoting substances, immunosuppressive substances, immunostimulatory substances, anti-allergic substances, anti-inflammatory substances, hematopoietic promoting substances, hormones, growth factors, enzymes, cytokines, chemocaines, lipid mediators, neurotransmitters, vitamins, minerals, antibacterial It can be a substance, an antiviral substance, an antifungal substance, an antiparasitic substance, or the like. Further, it may be a composition containing these substances.

The foreign substance may be, for example, an artificially synthesized compound. Such an artificially synthesized compound is preferable as an application target of the present invention because a desired amount of the compound can be obtained relatively easily. Such artificially synthesized compounds are derived from a compound created by using a substance derived from a natural resource as a lead substance, a compound obtained by artificially synthesizing a substance equivalent to a substance derived from a natural resource, and a natural resource. Examples thereof include compounds that are artificially designed and synthesized regardless of the substance. The method of synthesis is not particularly limited, and may be, for example, a substance synthesized by general chemical synthesis (typically organic synthesis) or biosynthesis (that is, production based on genetic engineering). Alternatively, the foreign substance may be a natural extract. For example, it can be a substance extracted (typically isolated) from a human or non-human organism.

Preferable examples of the foreign substance include, for example, amino acids, peptides, proteins, polynucleotides, viral particles, plasmids, vectors for gene transfer, lipids, sugars and the like.
Here, the peptides and proteins are not particularly limited as long as they are amino acid polymers having a plurality of polypeptide bonds. The number of amino acid residues contained in the peptide chain is not particularly limited. In general, peptides have a relatively small molecular weight and can, for example, have approximately 100 or less total amino acid residues (preferably 60 or less, for example 50 or less). Examples of such peptides and proteins include various antibodies, enzymes, hormones, cytokines, chemokines, cell surface factors and the like.
The polynucleotide is not particularly limited as long as it is a polymer (nucleic acid) in which a plurality of nucleotides are linked by a phosphodiester bond. The number of nucleotides constituting the polynucleotide is also not particularly limited, and may be either single-stranded or double-stranded. Further, it may have a linear shape, an annular shape, or an appropriately folded shape. For example, RNA fragments of various lengths (including mRNA, tRNA, microRNA, etc.), DNA fragments, and the like can be mentioned.
The amino acids, peptides, proteins, polynucleotides, virus particles, plasmids, gene transfer vectors, lipids, sugars and the like can be substances artificially synthesized by chemical synthesis or biosynthesis (that is, production based on genetic engineering). .. Alternatively, it may be a substance isolated from humans or non-human organisms.

Exosomes secreted from cells contain peptides (including proteins) and RNAs (eg, mRNA, microRNA, etc.) derived from the cells, and the inclusions are stably (typically degraded). Can be maintained (without). Therefore, peptides, proteins, and polynucleotides can be stably retained in exosomes for introducing foreign substances. In particular, these substances are susceptible to degradation by proteolytic enzymes (proteases), nucleolytic enzymes (nucleases) and the like in vivo (typically in blood) and elimination by immune cells. On the other hand, by encapsulating peptides, proteins, and polynucleotides in exosomes for introducing foreign substances, the stability of these substances in vivo can be improved. For the same reason, the present invention can be suitably applied to virus particles, plasmids, and vectors for gene transfer.

Further, the exosome for introducing a foreign substance disclosed here may preferably contain a highly hydrophobic substance as the foreign substance contained in the exosome.
Due to its low solubility, it may be difficult to supply a desired amount of such a highly hydrophobic substance into a cell culture medium. By encapsulating such a substance in an exosome for introducing a foreign substance, the supply amount of the hydrophobic substance to the cultured cells can be increased.
In addition, the highly hydrophobic substance is difficult to achieve a desired concentration in the living body (typically in blood), and is easily eliminated from the living body by metabolism and excretion. In particular, it tends to require administration at a high concentration (in the blood), administration at a high frequency, and the like. By encapsulating such a substance in an exosome for introducing a foreign substance, stability in a living body can be improved.

Alternatively, from the viewpoint of the medical industry, substances having antitumor activity such as doxorubicin (typically anticancer drugs) and substances having immunosuppressive activity such as tacrolimus hydrate (typically). Medical agents containing immunosuppressive substances) and the like, and metallic diagnostic agents such as magnetic beads and quantum dots are preferable as foreign substances contained in the exosomes for introducing foreign substances disclosed herein.

The method for incorporating the substance that induces macropinocytosis and the foreign substance into the exosome is not particularly limited, and examples thereof include an electroporation method (electroporation method). Specifically, a suspension in which exosomes and macropinocytosis inducers and / or foreign substances are suspended is prepared, and a predetermined electric pulse is applied to the suspension to impose minute amounts on the exosome membrane. By making a hole, the macropinocytosis inducer and / or foreign substance can be introduced into the exosome.

The macropinocytosis-inducing substance and the foreign substance may be simultaneously included (introduced) in the exosome, or may be separately included (introduced) in several times. From the viewpoint of operability and avoiding fusion of exosomes, it is preferable that the substance that induces macropinocytosis and the foreign substance are introduced into the exosome at the same time.

The ratio of exosomes, macropinocytosis-inducing substances and foreign substances suspended in the suspension depends on the type of substance to be introduced (encapsulated) into the exosome, the amount and composition of the substance to be included in the exosome, and the like. Can be set as appropriate. The ratio of exosomes to macropinocytosis-inducing substances in the suspension is: exosomes (g): substances that induce macropinocytosis (g) = 1: 0.1 to 1: 5 (preferably 1: 1). It can be 0.3 to 1: 3, more preferably 1: 0.5 to 1: 2, for example, about 1: 1). The ratio of exosomes to foreign substances (in the case of using two or more kinds of substances, the total mass of all foreign substances) in the suspension is exosome (g): foreign substance (g) = 1: 1. It can be 0.1 to 1:10 (preferably 1: 0.5 to 1: 8, more preferably 1: 1 to 1: 5, for example about 1: 2). Here, the content (g) of exosomes in the suspension is the content converted to the total protein amount of exosomes.
Typically, the higher the ratio of the content of macropinocytosis inducer in suspension to the content of exosome in suspension, the higher the concentration of macropinocytosis inducer in the exosome. Can be included in. Similarly, the higher the ratio (ratio) of the content of the foreign substance to the content of the exosome in the suspension, the higher the concentration of the foreign substance can be contained in the exosome. Also, typically, the proportion of macropinocytosis-inducing substances and foreign substances contained in exosomes is proportional to the proportion of macropinocytosis-inducing substances and foreign substances suspended in the suspension.

The composition (composition for introducing a foreign substance) used for introducing the foreign substance disclosed here from the outside of the cell into the cell of the target cell includes one or more kinds of foreign substances and macropinocytosis. It is a composition containing an exosome containing a substance that induces. That is, the composition for introducing a foreign substance is a composition containing the above-mentioned exosome for introducing a foreign substance.

The composition for introducing a foreign substance disclosed herein depends on the mode of use as long as the exosome and the macropinocytosis inducer can be retained in a state in which the foreign substance contained in the exosome can be introduced into the target cell. It may contain a variety of pharmaceutically acceptable carriers. That is, the composition for introducing a foreign substance disclosed here is a composition that holds a foreign substance and a macropinocytosis-inducing substance in a state of being encapsulated in an exosome without losing their activities. As the carrier, a carrier generally used in pharmaceutical products as a diluent, an excipient, or the like is preferable. Although it may vary depending on the use and form of the composition for introducing a foreign substance, water, a physiological buffer solution, and various organic solvents are typically used. It can be an aqueous solution of an alcohol (such as ethanol) of appropriate concentration, a non-drying oil such as glycerol, olive oil. Alternatively, it may be a liposome. In addition, examples of secondary components that can be contained in the composition for introducing foreign substances include various fillers, bulking agents, binders, wetting agents, surfactants, pigments, fragrances and the like.

Alternatively, the composition for introducing a foreign substance disclosed herein is a composition containing an exosome containing one or more kinds of foreign substances and a substance that induces macropinocytosis in a target cell. could be. As long as the foreign substance-introducing composition can retain the foreign substance contained in the exosome and the macropinocytosis-inducing substance in a state in which these activities are not lost, the composition is pharmaceutical (pharmaceutical) depending on the mode of use. ) It may contain various carriers that are acceptable. As the carrier, those as described above can be used without particular limitation.

There is no particular limitation on the form of the composition for introducing a foreign substance. For example, typical forms include liquids, suspensions, emulsions, aerosols, foams, granules, powders, tablets, capsules, ointments, aqueous gels and the like. It can also be used as a lyophilized product or granulated product for preparing a drug solution by dissolving it in physiological saline or an appropriate buffer solution (for example, Phosphate-Buffered Saline (PBS)) immediately before use.
In addition, exosomes containing foreign substances and substances that induce macropinocytosis, or exosomes (main components) containing foreign substances and substances that induce macropinocytosis, and various carriers (secondary components) are used as materials. The process itself for preparing various forms of the drug (composition) may be based on a conventionally known method, and the formulation method itself does not characterize the present invention, and thus detailed description thereof will be omitted. Detailed sources of prescribing information include, for example, Comprehensive Medicinal Chemistry, supervised by Corwin Hansch, published by Pergamon Press (1990). The entire contents of this book are incorporated herein by reference.

The cells to which the exosome for introducing a foreign substance and the composition for introducing a foreign substance disclosed herein are applied are not particularly limited, and the foreign substance can be introduced into the cells into various cells. For example, cells of humans or non-human animals (typically vertebrates, especially mammals). As the application target cells of the exosome for introducing a foreign substance and the composition for introducing a foreign substance disclosed herein, human cells (cells derived from humans) are particularly preferable.
For example, it may be a cell (derived from various tissues in the living body) existing in various tissues in the living body such as epithelial tissue, connective tissue, muscle tissue, and nerve tissue. The target cells are not limited to normal cells, but may be cells that cause diseases such as tumor cells and pathogen-infected cells. In addition, the differentiation state of the target cells is not particularly limited, and foreign substances can be suitably introduced into cells at various differentiation stages such as mature somatic cells, progenitor cells, somatic stem cells, and pluripotent stem cells. can.
The application target cells of the exosome for introducing a foreign substance and the composition for introducing a foreign substance are not limited to cultured cells maintained in vitro, but can also be applied to cells existing in vivo.

The cells to which the exosome for introducing a foreign substance and the composition for introducing a foreign substance disclosed herein are applied are included in the exosome for introducing a foreign substance and the composition for introducing a foreign substance to be used (typically, contained in the exosome). ) Cells in which macropinocytosis is induced by a macropinocytosis inducer are preferred. Typically, when a substance that induces macropinocytosis by stimulating a specific receptor is used as a macropinocytosis inducer, the receptor stimulated by the macropinocytosis inducer is expressed. Cells that are (typically highly expressed) are preferred. For example, when a substance that stimulates EGFR is used as the macropinocytosis inducer, EGFR-expressing cells (more preferably, EGFR-highly expressing cells) are preferable as the target cells. Further, for example, when a substance that stimulates CXCR4 is used as the macropinocytosis inducer, CXCR4-expressing cells (more preferably CXCR4-highly expressing cells) are preferable as the target cells. Similarly, when a substance that stimulates PDGFR is used as the macropinocytosis inducer, PDGFR-expressing cells (more preferably PDGFR-expressing cells) are preferable as the target cells, and FGFR is used as the macropinocytosis-inducing substance. And / or if a substance that stimulates Syndecan-4 is used, cells expressing at least one of FGFR and Syndecan-4 (more preferably cells expressing both FGFR and Syndecan-4) are targeted. Preferred as a cell. As a result, foreign substances can be introduced into the target cells with high efficiency.

When the exosome for introducing a foreign substance and the composition for introducing a foreign substance disclosed herein are applied to cultured cells, the cultured cells to be applied are not particularly limited. For example, it can be various cultured cells such as primary cultured cells, passaged cells, and cell lines (cell lines) derived from humans or non-human animals (typically mammals).

The exosomes for introducing foreign substances and the compositions for introducing foreign substances disclosed herein can be used in methods and doses according to their forms and purposes.
For example, in the case of introducing a foreign substance into cells cultured (passage) in vitro (in vitro), an appropriate amount of the exosome for introducing a foreign substance or the composition for introducing a foreign substance disclosed here is used. , The target cells may be added to the medium at any stage of the culturing process (for example, at an early stage after the start of culturing, or after culturing (proliferation) or subculture for a predetermined period). The amount and number of additions are not particularly limited because they may vary depending on conditions such as the type of cultured cells, cell density (cell density at the start of culture), number of passages, culture conditions, type of medium, and the like. Typically, the exosome concentration in the medium is generally in the range of 0.1 μg / mL to 100 μg / mL, preferably 0.2 μg / mL to 50 μg / mL (eg 0.4 μg / mL to 40 μg / mL). It is preferable to add the exosome for introducing a foreign substance and the composition for introducing a foreign substance one or more times (for example, additional addition at the start of culture and at the time of cell passage or medium exchange) so as to be within the range. ..
In addition, the exosome for introducing a foreign substance and the composition for introducing a foreign substance disclosed herein should be used in combination with a substance having a physiological activity (typically a substance having a pharmacological activity) other than the foreign substance contained in the exosome. Is also possible.

Alternatively, the exosome for introducing a foreign substance and the composition for introducing a foreign substance disclosed herein can be supplied to a patient (that is, a living body) in a desired amount. The administration method is not particularly limited. Examples thereof include intravenous, intraarterial, intradermal, subcutaneous, intraperitoneal injection, oral administration, inhalation administration, transdermal administration, transmucosal administration, suppository administration and the like. Alternatively, it may be administered as an implantable preparation by implanting it subcutaneously, subfascial, or the like.

The organism to which the exosome for introducing a foreign substance and the composition for introducing a foreign substance is administered may be a human or an animal other than a human (typically a mammal). The use of exosomes for introducing foreign substances or compositions for introducing foreign substances in humans is particularly preferable because of its high value in the medical industry. In addition, it is preferable to target test animals such as mice, rats, guinea pigs, rabbits, dogs, and cynomolgus monkeys from the viewpoint of promoting various research and development. In addition, it is preferable to target pet animals such as dogs and cats because of their high utility value in the veterinary medical industry.

When an exosome for introducing a foreign substance and a composition for introducing a foreign substance are administered into a living body, it is strongly required to ensure the safety of the exosome and the composition and to reduce the immune response.
From the viewpoint of safety against foreign substance-introducing exosomes (for example, cell toxicity by foreign substance-introducing exosomes), cells into which foreign substances are introduced using the exosomes (hereinafter, the cells are also referred to as "recipient cells"). It is preferable that the species is the same as the species of the organism or cell that collects the exosome (hereinafter, the organism is also referred to as a "donor organism" and the cell is also referred to as a "donor cell"). For example, when introducing a foreign substance into a human-derived cell, it is preferable to use a human (or human-derived cell) -derived exosome. Further, it is preferable that the species of the recipient cell and the donor cell (donor organism) are the same from the viewpoint of efficiently introducing the exosome for introducing a foreign substance into the target cell (recipient cell).
From the viewpoint of reducing the immune response to exosomes for introducing foreign substances, it is preferable that the human leukocyte antigen (HLA) type of the recipient cell and the HLA type of the donor cell (donor organism) are the same. .. Alternatively, it is preferred that the donor cell (donor organism) is a homodonor of one of the two HLAs possessed by the recipient cell. Thereby, it is possible to reduce (avoid) the immune reaction (immune rejection reaction) to the exosome for introducing a foreign substance in the living body into which the exosome for introducing a foreign substance is introduced. From the viewpoint of reducing the immune response to a higher degree, it is preferable that the recipient cell and the donor cell (donor organism) are derived from the same living body (individual). In other words, exosomes for introducing foreign substances are recovered from living organisms (typically patients) to which foreign substances are to be introduced using exosomes for introducing foreign substances, and the exosomes are used for exosomes for introducing foreign substances (compositions for introducing foreign substances). Is preferable.

According to the technique disclosed here, an exosome for introducing a foreign substance (composition for introducing a foreign substance) containing a substance having antitumor activity (typically an anticancer drug) as a foreign substance is introduced into the living body of a tumor patient. By administration, the substance having antitumor activity can be suitably introduced into tumor cells. That is, by using a substance having antitumor activity as a foreign substance, the exosome for introducing a foreign substance (composition for introducing a foreign substance) according to the present invention can be used for the purpose of treating a tumor (cancer).
Further, the exosome for introducing a foreign substance (composition for introducing a foreign substance) disclosed here can be suitably used for a drug delivery system for introducing a substance having antitumor activity into tumor cells. Further, according to the technique disclosed here, a method for treating a tumor, in which an exosome for introducing a foreign substance (composition for introducing a foreign substance) containing a substance having antitumor activity as a foreign substance is administered in vivo. Can be provided.

Tumor cells are often in a state in which macropinocytosis is likely to be induced because extracellular nutrients are taken up into the cells. Typically, tumor cells often highly express EGFR, CXCR, PDGFR, and / or FGFR. Therefore, by using substances that stimulate these receptors (typically EGF, SDF-1, PDGF, or FGF) as macropinocytosis inducers, tumor cells (typically EGFR, CXCR, Macropinocytosis of PDGFR and / or tumor cells highly expressing FGFR) can be induced at high levels. As a result, tumor cells (typically tumor cells that highly express EGFR, CXCR, PDGFR and / or FGFR) have an antitumor effect due to a foreign substance (here, a substance having antitumor activity) contained in the exosome. ) Can be selectively exerted.

In addition, exosome particles having a diameter of about 50 nm to 100 nm have a property of easily accumulating in tumor tissue due to an EPR effect (enhanced permeability and retention effect). Therefore, the antitumor effect of the foreign substance contained in the exosome (here, the substance having antitumor activity) can be selectively exerted on the tumor cells. Here, the EPR effect is because (I) there is a gap of about several hundred nm in the tumor tissue (typically a growing tumor tissue) and the blood vessel wall (vascular endothelium) of the new blood vessels surrounding it. In addition, particles having a particle size of several tens of nm to 200 nm (particularly about 100 nm) easily infiltrate (permeate) around the tumor tissue, and (II) the lymphatic tissue is often immature. It refers to the characteristic that a substance of a specific size easily accumulates around a tumor cell because the infiltrated (permeated) substance is not eliminated and easily accumulates.
For the reasons described above, an exosome for introducing a foreign substance (composition for introducing a foreign substance) containing a substance having antitumor activity according to the present invention is supplied to a tumor patient (typically administered in vivo). By doing so, it is possible to exert a high level of antitumor effect while reducing side effects on normal cells.

When the exosome for introducing a foreign substance according to the present invention is applied to a tumor cell, it is particularly effective for a foreign substance having a particle size of less than 20 nm (for example, 5 nm or less, typically 1 nm or less, particularly 0.5 nm or less). The present invention can be preferably applied. Since a substance having such a particle size has a relatively smaller particle size than a substance that easily infiltrates (permeates) around tumor cells due to the EPR effect, it is difficult for the EPR effect to be exhibited (that is, tumor tissue-specific accumulation (introduction)). It is a substance (difficult to do). By encapsulating such a substance having a relatively small particle size in an exosome, it is possible to obtain a particle size that easily accumulates in a tumor tissue-specific manner due to the EPR effect. This makes it possible to exert the antitumor effect of such a foreign substance in a tumor cell-specific manner.

Alternatively, for example, a stem cell (for example, a large number of exosomes for introducing a foreign substance (or a composition for introducing a foreign substance containing the exosome) containing a substance having differentiation-inducing activity as a foreign substance (typically, a differentiation-inducing agent)). By applying to capable stem cells, somatic stem cells, progenitor cells, etc.), the exosome (or a composition for introducing a foreign substance containing the exosome) can be used for regenerative medicine. Typically, a differentiation-inducing substance is introduced into stem cells cultured (passaged) in vitro (in vitro) by using an exosome for introducing a foreign substance (composition for introducing a foreign substance) according to the present invention. By doing so, the stem cells can be differentiated into desired cells. Thereby, a desired cell or the like, typically a differentiated cell (including a cell mass, a tissue, an organ, etc.) and a biosynthesis derived from the cell can be produced (produced). Such differentiated cells and the like can be suitably used as materials for regenerative medicine (typically, cell resources for transplantation). For example, by returning such differentiated cells or the like to the affected area where repair or regeneration is required (that is, in the living body of the patient), the repair or regeneration can be effectively performed. That is, it becomes possible to efficiently treat various diseases in which tissue regeneration is a promising treatment method. Here, examples of biosynthesis derived from differentiated cells produced by inducing differentiation from the stem cells include physiologically active substances such as secretory proteins and hormones (for example, insulin).

Hereinafter, some examples of the present invention will be described, but the present invention is not intended to be limited to those shown in such examples.

<Test 1: Preparation of cell culture (maintenance of passage of cells to be evaluated)>
Here, HeLa cells (derived from human cervical cancer), A431 cells (derived from human epidermal cancer), MIA PaCa-2 cells (derived from human pancreatic cancer) and BxPC-3 cells (derived from human pancreatic cancer) shown in Table 1 are used. An evaluation test was performed using it. All of these four types of cells are human-derived cultured cell lines. As these evaluation target cells, those that were subcultured as follows were used.
Specifically, each cell was seeded in a culture dish (100 mm dish) having a diameter of 100 mm and containing 10 mL of a medium for subculture, and allowed to stand in an incubator under the conditions of _{5% CO 2 and 37 ° C.} It was cultured. Here, as the subculture medium, those having the compositions shown in Table 1 were used. The "medium" column in Table 1 indicates the type of medium used for subculture of each cell, and the "additive" column in Table 1 indicates "additives" in Table 1 according to each cell. The substance added to the medium shown in the "medium" column is shown, and the content of the "additive" contained in the subculture medium is shown in the "content" column in Table 1. In Table 1, (G) is "manufactured by Life Technologies (Gibco)", (H) is "manufactured by Thermo Scientific (Hyclone)", and (S) is "manufactured by Sigma-Aldrich". , And (W) indicates that it is "manufactured by Wako". Hereinafter, unless otherwise specified, such a medium is used for culturing each cell.
In addition, all of the above-mentioned target cells were passaged once every 2 to 3 days. For cell detachment, trypsin (0.1 g / L) -millylenediaminetetraacetic acid (EDTA) (0.11 mmol / L) (manufactured by Nacalai Tesque) was added to each culture dish at a rate of 2 mL / well and allowed to react at 37 ° C. for 5 minutes. went.

<Test 2: Preparation of exosome-producing cells>
By the following method, a fusion protein in which CD63, which is a marker protein of exosome, and green fluorescent protein (GFP) are fused (hereinafter, such fusion protein is also simply referred to as “CD63-GFP”) is stably expressed. Established a cell line to be used. Since the exosomes recovered from such a cell line are fluorescently colored green by GFP fused to CD63, the presence of exosomes can be confirmed by observing the fluorescent coloration of the GFP. Here, the CD63-GFP expression gene was introduced into cells by the lipofection method, and the cells into which the genes were introduced were selected with an appropriate drug to establish the CD63-GFP stable expression cell line. Specifically, it is as follows.

First, pCT-CD63-GFP manufactured by System Biosciences was purchased and prepared as a CD63-GFP expression gene. Such pCT-CD63-GFP is obtained by loading (inserting) a cytomegalovirus (CMV) promoter, a gene encoding CD63-GFP, and a puromycin resistance gene into a lentivirus vector. In addition, as a lipofection reagent used in the above lipofection method, Lipofectamine LTX Reagent manufactured by Life Technologies (Invitrogen) was purchased and prepared.
Next, HeLa cells were seeded in a 24-well culture dish (24-well dish) to ^{a cell density of 4.7 × 10 4} cells / well, and in an incubator under the conditions of _{5% CO 2, 37 ° C.} Was cultured for 1 day. As the medium, a medium having the same composition as that used as the subculture medium for HeLa cells in Test 1 above (hereinafter, such a medium is also referred to as “HeLa medium”) was used so as to be 1.0 mL per well. ..

Then, for the HeLa cells after culturing for the above 1 day, the medium in each culture vessel was replaced with 200 μL of HeLa medium containing a complex of pCT-CD63-GFP and Lipofectamine LTX Reagent, and 5% CO ₂ , 37 ° C. The CD63-GFP expression gene was transfected into cells by culturing in an incubator under the conditions for 1 day. The above complex contains pCT-CD63-GFP and Lipofectamine LTX Reagent in a mixed solution with a pCT-CD63-GFP content of 800 ng / well and a Lipofectamine LTX Reagent content of 1 according to the manual attached to the Lipofectamine LTX Reagent. It was prepared by mixing so as to have a volume% (1% (v / v)).
Next, the cells after the transfection were subcultured in 1 mL of HeLa medium containing 3 μg / mL of puromycin (Pyuromycin, manufactured by Nacalai Tesque) (hereinafter, such medium is also referred to as “p-HeLa medium”). .. As a result, cells that stably express CD63-GFP (hereinafter, such cells are also referred to as "CD63-GFP-HeLa") were established.

<Test 3: Recovery of exosomes>
Using the CD63-GFP-HeLa cells prepared by the method described above, the exosomes secreted by the cells into the medium were recovered. Here, the ultracentrifugation method (Thery C., Curr. Protoc. Cell Biol. (2006) Chapter 3: Unit 3.22.) Or Total exosome isolation (from cell culture media) (manufactured by Life Technologies (Invitrogen)) is used. The exosomes in the medium were separated and recovered using. Specifically, it is as follows.

First, the above CD63-GFP-HeLa cells were seeded in a culture dish (100 mm dish) having a diameter of 10 cm at a cell density of ^{2 × 10 6} cells / well, and 1 in an incubator under the conditions of _{5% CO 2, 37 ° C.} Incubated for days. As the medium, 10 mL of the above p-HeLa medium was used per well.
For the CD63-GFP-HeLa cells after culturing for the above 1 day, the medium in the culture vessel was removed, and the inside of the culture vessel was washed 5 times with α-MEM medium (5 mL). This allows exosomes that may be contained in the medium (typically FBS) to be removed from the culture vessel. Then, 1% of α-MEM medium (manufactured by Life Technologies (Gibco)) containing 10% of FBS (Exosome-depleted FBS Media Supplement, manufactured by System Biosciences) containing no exosomes and 3 μg / mL of puromycin. 10 mL was added per well, and the cells were _{cultured under the conditions of 5% CO 2} , 37 ° C. for 3 to 4 days. Then, after culturing for the above predetermined period, the medium in the culture vessel was collected.

Next, the exosomes secreted in the collected medium were separated and recovered by an ultracentrifugal method. Specifically, after centrifuging at 300 × g at 4 ° C for 10 minutes, the supernatant is centrifuged at 2,000 × g at 4 ° C for 10 minutes, and then the supernatant is further prepared. A series of centrifugation operations were carried out by centrifuging at 10,000 × g and 4 ° C. for 30 minutes to precipitate dead cells and the like that could float in the medium. The supernatant after performing such a centrifugation operation was collected, and the supernatant was centrifuged at 100,000 × g at 4 ° C. for 70 minutes twice to precipitate exosomes. Then, by collecting the precipitate, exosomes secreted by CD63-GFP-HeLa cells into the medium (hereinafter, such exosomes are also referred to as "CD63-GFP-exosomes") were collected. The obtained exosomes were dispersed in DPBS (Dulbecco's Phosphate-Buffered Saline, manufactured by Nacalai Tesque) to prepare an exosome stock solution.

Alternatively, Total exosome isolation (from cell culture media) (manufactured by Life Technologies (Invitrogen)) was used to separate and recover exosomes secreted into the medium recovered after culturing for the above predetermined period. The above Total exosome isolation (from cell culture media) is a commercially available kit for isolating exosomes from cell culture medium, and the specific operation was performed according to the manual attached to the kit.

The concentration of recovered exosomes was quantified using a BCA protein assay kit (manufactured by Thermo Scientific (Pierce)). Hereinafter, the protein concentration (μg / mL) obtained by such quantification will be treated as the exosome concentration (μg / mL). The presence and shape of exosomes were confirmed by detection of exosome marker proteins (CD63, CD9, etc.) by Western blotting and observation using a transmission electron microscope (negative staining).

<Test 4: Introduction of foreign substances into exosomes (inclusion)>
A foreign substance and a substance that induces macropinocytosis were introduced into the exosome (CD63-GFP-exosome) obtained in Test 3 above. Saporin (manufactured by Sigma-Aldrich, the same applies hereinafter) was used as the foreign substance, and EGF (manufactured by Sigma-Aldrich, the same applies hereinafter) was used as the substance that induces macropinocytosis. As a comparative example, transferrin (manufactured by Sigma-Aldrich, the same applies hereinafter) known to induce clathrin-dependent endocytosis was encapsulated in exosomes. The electroporation method was adopted as a method for introducing these substances into exosomes. The details of the test are as follows.
A dispersion was prepared in which 25 μg of CD63-GFP-exosome and 50 μg of saporin and / or 25 μg of EGF were dispersed in 100 μL of DPBS. Alternatively, a dispersion in which 25 μg of CD63-GFP-exosome and 25 μg of transferrin were dispersed in 100 μL of DPBS was prepared. Then, the dispersion was electroporated using Super electropolator NEPA21 Type II manufactured by NEPA genes. Electroporation was performed under the following conditions: poring pulse: twice pulse, 200 V, 5 msec; transfer pulse: five pulse, 20 V, 50 msec; 1 cm electroporation cuvette; 25 ° C :.
The solution after electroporation was filtered through Amicon Ultra Filtration filters (100 kDa) manufactured by Merck Millipore to remove saporins, EGF, and transferrin that were not introduced (encapsulated) into exosomes. Here, the above filtration was carried out by centrifugation for 10 minutes under the condition of 18,000 × g and 4 ° C., and the exosomes remaining on the filter were collected. Then, the exosome after such filtration was dispersed in 500 μL of DPBS, and the washing operation of 18,000 × g and centrifugation for 10 minutes under the condition of 4 ° C. was repeated twice. As described above, exosomes containing saporin and / or EGF, or exosomes containing transferrin were prepared. Such exosomes were dispersed in DPBS to prepare an exosome stock solution.

Hereinafter, the CD63-GFP-exosome obtained in the above test 3 is sample 1, the saporin-encapsulating exosome obtained in the above test 4 is sample 2, the EGF-encapsulating exosome obtained in the above test 4 is sample 3, and the above test 4 is used. Sample 4 is the exosome containing EGF and saporin obtained, and sample 5 is the exosome containing transferrin obtained in Test 4 above. Table 2 summarizes the exosomes of Samples 1-5.

<Test 5: Exosome uptake test>
A confocal microscope was used to capture the exosomes of the test cells when the exosomes of Sample 1 and EGF as a substance that induces macropinocytosis were supplied into the medium of the test cells. It was evaluated by fluorescence observation. A431 cells were used as test cells. The details of the test are as follows.

First, A431 cells were seeded in a glass-based dish having a diameter of 35 mm at ^{a cell density of 2.0 × 10 5} cells / well, and precultured for 1 day in an incubator under the conditions of _{5% CO 2 and 37 ° C.} .. As the medium, 2 mL of a medium having the same composition as that used as the subculture medium for A431 in Test 1 (hereinafter, such a medium is also referred to as “A431 medium”) was used.
For A431 cells after the above-mentioned 1-day culture (preculture), the operation of removing the medium in the culture vessel and washing the inside of the culture vessel with 1 mL of A431 medium (that is, MEM medium) containing no FBS was performed three times. Repeated. Then, 20 μg / mL of exosomes according to Sample 1 and MEM medium containing 500 nM of EGF were added in an amount of 200 μL per well, and the cells were _{cultured under the conditions of 5% CO 2} and 37 ° C. for 24 hours. Further, as a control group, a test group (EGF-free group) was provided in which the exosome according to Sample 1 of 20 μg / mL was added and EGF was not added.

For the cells after culturing in the presence of the above exosomes, Hoechst33342 (manufactured by Life Technologies (Invitrogen)) in an amount of 5 μg / mL was added to the culture medium, and the cells were left to stand for 15 minutes under a temperature condition of 37 ° C. Stained the nucleus. The cells after such nuclear staining were washed 3 times with the above A431 medium (1 mL). Then, 1 mL of A431 medium was added to the culture vessel, and fluorescence observation was performed using a confocal microscope without fixing the target cells (that is, in the state of living cells). As a measuring device, FV1200 manufactured by Olympus was used.

FIG. 1 shows the results of fluorescence observation with a confocal microscope (confocal laser microscope) for each test group. These images are micrographs (images) of the intracellular uptake of exosomes according to Sample 1 in each test group. Specifically, the photograph (image) shown in the left column (CD63-GFP-exosome column) is a micrograph in which the localization of exosomes related to sample 1 is investigated. In the photograph (image) shown in the right column (Merge column), the image of nuclear staining by Hoechst33342 and the image of examining the localization of exosomes related to sample 1 shown in the left column were superimposed (merged). ) It is an image.

As shown in FIG. 1, the test group in which EGF was added and cultured (EGF-added group, lower part of FIG. 1) had stronger fluorescence color development of GFP as compared with the EGF-free group (upper part of FIG. 1). .. This indicates that the EGF-added group has more active exosome uptake than the EGF-free group. From the above results, it was confirmed that by supplying EGF to the target cells, the uptake of exosomes into the cells by the cells can be promoted.

<Test 6: Exosome uptake test>
When the exosome according to the above sample 1 and EGF as a substance for inducing macropinocytosis were supplied into the medium of the test cell, the uptake of the exosome by the test cell was analyzed using a flow cytometer. evaluated. A431 cells were used as test cells. The details of the test are as follows.
First, A431 cells were seeded in a 24-well culture dish (24-well plate) at a cell density of ^{4.7 × 10 4} cells / well, and pre-cultured for 1 day in an incubator under the conditions of _{5% CO 2 and 37 ° C.} Was done. As the medium, 1.0 mL of A431 medium was used per well.
For A431 cells after the above-mentioned 1-day culture (preculture), the medium in the culture vessel was removed, and the inside of the culture vessel was washed 3 times with MEM medium (200 μL). Then, 200 μL of the medium having the composition shown in Table 3 was added per well, and the cells were _{cultured under the conditions of 5% CO 2} and 37 ° C. for 24 hours. Specifically, in Test Group 6-1 to Test Group 6-3, MEM medium (that is, A431 medium containing no FBS) containing EGF at the concentration shown in Table 3 and exosomes related to Sample 1 was used. .. In addition, Test Group 6-4 and Test Group 6-5 used A431 medium (that is, MEM medium containing 10% FBS) containing EGF and exosomes according to Sample 1 at the concentrations shown in Table 3.

The cells after culturing in the presence of the above exosomes were washed 3 times with DPBS (200 μL). Then, trypsin (0.1 g / L) -ethylenediaminetetraacetic acid (EDTA) (0.11 mmol / L) (manufactured by Nacalai Tesque) was added to the culture vessel at a rate of 200 μL / well, and the cells were allowed to react at 37 ° C. for 10 minutes to exfoliate the cells. bottom. The detached cells were collected in a centrifuge tube, centrifuged at 800 × g at 4 ° C. for 5 minutes, and then the supernatant was removed. Then, 400 μL of DPBS was added to each centrifuge tube to suspend the cells in the DPBS, and the cells were centrifuged again at 800 × g at 4 ° C. for 5 minutes, and the supernatant was removed. Then, the cells of each test group were suspended in 400 μL of DPBS.
The cell suspensions of each test plot prepared as described above were analyzed using a flow cytometer. As a measuring device, Guava easy Cyte manufactured by Merck Millipore was used. Specifically, the average GFP fluorescence intensity per cell in each test was measured by detecting the fluorescence color development of GFP under the conditions of an excitation wavelength: 488 nm and an emission wavelength: 525 nm.

The results of the analysis using the flow cytometer as described above are shown in FIG. The graph shown in FIG. 2 shows the relative value of the average GFP fluorescence intensity per cell in each test group when the average GFP fluorescence intensity per cell in the test group 6-1 (control group) is 100. be. The bar graph shown in FIG. 2 shows the results of test plots 6-1 and 6-2, 6-3, 6-4, and 6-5 in order from the left.

As shown in FIG. 2, the average GFP fluorescence intensity per cell in Test Group 6-2 and Test Group 6-3 is remarkable as compared with the average GFP fluorescence intensity per cell shown in Test Group 6-1. It was expensive. In addition, the average GFP fluorescence intensity per cell increased as the concentration of EGF in the medium increased (that is, depending on the concentration of EGF in the medium). Specifically, the average GFP fluorescence intensity per cell in Test Group 6-2 is about 19 times that of the average GFP fluorescence intensity per cell in Test Group 6-1. The average GFP fluorescence intensity per cell was approximately 27-fold. From the above results, it was confirmed that the uptake of exosomes into cells is remarkably promoted by supplying EGF to the target cells.

Further, as shown in FIG. 2, the average GFP fluorescence intensity per cell in Test Group 6-5 was compared with the average GFP fluorescence intensity per cell in Test Group 6-1 and Test Group 6-4. It was significantly higher. Specifically, the average GFP fluorescence intensity per cell in Test Group 6-5 was approximately 33 times that of the average GFP fluorescence intensity per cell in Test Group 6-1 and was found in Test Group 6-4. It was about 5 times the average GFP fluorescence intensity per cell. That is, from the results of Test Groups 6-1 to 6-5, it was confirmed that supplying EGF to the target cells can promote the uptake of exosomes into the cells regardless of the presence or absence of FBS in the medium. ..
In Test Groups 6-4 and 6-5, since the exosomes derived from FBS are contained in the medium, the exosomes derived from FBS and the exosomes related to Sample 1 are in a competitive state. Such a competitive state is that when an exosome for introducing a foreign substance (composition for introducing a foreign substance) according to the present invention is supplied (administered) into a living body, an endogenous exosome (exosome originally present in the living body) and a foreign substance are exhibited. This is a reproduction of the state of competition with the introduction exosome in an in vitro experimental system. That is, from the results of Test Groups 6-4 and 6-5, even in an environment where endogenous exosomes (here, FBS-derived exosomes) are present, the target exosomes can be supplied by supplying EGF to the target cells. It was confirmed that (that is, exosomes for introducing foreign substances) can be remarkably promoted into the cells.

<Test 7: Evaluation test of macropinocytosis-inducing activity by EGF>
Whether the intracellular uptake of exosomes promoted by supplying EGF to the target cells was due to macropinocytosis was evaluated by fluorescence observation using a confocal microscope. Specifically, the intracellular localization of the marker substance that identifies the uptake of the substance by macropinocytosis and the intracellular localization of the exosome according to Sample 1 are observed by fluorescence using a confocal microscope. Compared. In this test, dextran (a polysaccharide of about 70 kDa) known to be taken up into cells by macropinocytosis was used as a marker substance for identifying the uptake of the substance by macropinocytosis. As the test cells, A431 cells were used. The details of the test are as follows.

Specifically, when culturing in the presence of exosomes, except that a medium containing EGF (500 nM) and exosomes (20 μg / mL) according to Sample 1 and dextran having a concentration of 0.5 mg / mL was used. The analysis was performed under the same conditions and methods as in Test 5. Here, as the dextran, a dextran labeled with a fluorescent dye (Texas red) (Texas red-dextran, manufactured by Life Technologies (Molecular Probes), the same applies hereinafter) was used.

FIG. 3 shows the results of fluorescence observation with a confocal microscope (confocal laser microscope). These images are micrographs (images) of the intracellular uptake of exosomes and Texas red-dextran according to Sample 1 by the test cells in this test. Specifically, the photograph (image) shown in the upper left is a micrograph in which the localization of exosomes according to Sample 1 is investigated. The photograph (image) shown in the lower left is an image showing the localization of Texas red-dextran. In the photograph (image) shown on the right, an image of examining the localization of exosomes related to sample 1 shown in the upper left of the above and an image of examining the localization of Texas red-dextran shown in the lower left of the above were superimposed (merged). It is an image.

As shown in FIG. 3, the intracellular localization of the exosome according to Sample 1 and the intracellular localization of dextran overlapped. That is, the exosome and dextran according to Sample 1 were co-localized in the cell. This indicates that the mechanism of uptake of exosomes and dextran into cells is the same, that is, exosomes and dextran are taken up into cells by macropinocytosis. From the above results, it was confirmed that the intracellular uptake of exosomes promoted by supplying EGF to the target cells was due to macropinocytosis. In other words, it was confirmed that EGF induces macropinocytosis, which promotes the intracellular uptake of exosomes. That is, it was confirmed that EGF can be suitably used as a substance that induces macropinocytosis.

<Test 8: Evaluation test of macropinocytosis-inducing activity by EGF>
Whether the intracellular uptake of exosomes promoted by supplying EGF to the target cells was due to macropinocytosis was evaluated by analysis using a flow cytometer. Specifically, the effects of macropinocytosis inhibitors on exosome uptake were analyzed using a flow cytometer. In this test, EIPA (5- (N-ethyl-N-isopropyl) amiloride, manufactured by Sigma-Aldrich) was used as the macropinocytosis inhibitor. A431 cells were used as test cells. The details of the test are as follows.

First, A431 cells pre-cultured for 1 day were prepared under the same conditions and methods as in Test 6 above, and the cells were washed 3 times using MEM medium. Then, 200 μL of MEM medium containing 100 nM EIPA was added per well, and the medium was left for 30 minutes under the conditions of _{5% CO 2 and 37 ° C.} Next, in the MEM medium containing EIPA, an amount of EGF having a concentration of 100 nM in the medium and an exosome according to Sample 1 having an amount of a concentration of 20 μg / mL in the medium were added, and 5% CO ₂ was added. The cells were cultured at 37 ° C. for 3 hours. Such a test plot is called an EIPA treatment plot. As a control, a test group (EIPA untreated group) was provided, which was treated in the same manner as the EIPA test group except that the above EIPA was not added to the MEM medium.
The cells after culturing in the presence of the exosomes were analyzed using a flow cytometer under the same conditions and methods as in Test 6 above.

The results are shown in FIG. The graph shown in FIG. 4 shows the relative value of the average GFP fluorescence intensity per cell in the EIPA-treated group when the average GFP fluorescence intensity per cell in the EIPA-untreated group is 100.

As shown in FIG. 4, the average GFP fluorescence intensity per cell in the EIPA-treated group was significantly lower than the average GFP fluorescence intensity per cell in the EIPA-untreated group (approximately 50% or less). ). This indicates that the uptake of exosomes by the target cells was inhibited by adding and culturing EIPA, which is an inhibitor of macropinocytosis. That is, it is shown that EGFA inhibited the macropinocytosis-inducing activity by EGF. From the above results, it was confirmed that the intracellular uptake of exosomes promoted by supplying EGF to the target cells was due to macropinocytosis. In other words, it was confirmed that by supplying EGF to the target cells, macropinocytosis of the cells was induced, which promoted the uptake of exosomes in the cells. That is, it was confirmed that EGF can be suitably used as a substance that induces macropinocytosis.

<Test 9: Evaluation test of cell viability>
Regarding the cell viability when the exosomes according to Sample 1 were supplied to the test cells and cultured, WST-1 (4- [3- (4-iodophenyl) -2- (4-nitrophenyl) -2H-5-tetrazolio) ] -1,3-benzene disulfonate) (water-soluble tetrazolium salt) was evaluated by performing a cell proliferation test (WST-1 assay) by absorptiometry. A431 cells were used as test cells. The details of the evaluation test are as follows.

A431 cells were seeded in each well of a 96-well plate so that the number of cells was approximately 1.2 × 10 ⁴ per well, and cultured for 1 day in an incubator under the conditions of _{5% CO 2 and 37 ° C.} (Preculture) was performed. As the medium, the above A431 medium was used, and the amount of medium per well was 100 μL.
After culturing (preculture) for the above predetermined time, the medium in the culture vessel is a MEM medium containing 20 μg / mL exosomes according to Sample 1 and 0 nM (that is, EGF-free), 100 nM or 500 nM EGF (that is, EGF). , FBS-free A431 medium). The amount of medium per well was 50 μL. As a control group, a test group containing neither exosome nor EGF related to sample 1 was provided in the medium.

After supplying the cells with exosomes as described above, a 96-well plate _{was placed in a CO 2} incubator and main-cultured under 37 ° C. and 5% CO _{2 conditions.} Then, when 24 hours have passed from the start of the culture (main culture) in the presence of exosomes, the viable state (number of living cells) of the test cells is measured with a commercially available color development measurement kit (Premix WST-1 Cell Proliferation Assay System, Takara). Measured using Bio). That is, by utilizing the fact that the tetrazolium salt in the reagent is reduced by the enzymatic activity of living cells to produce water-soluble formazan, the amount of water-soluble formazan in the medium is measured by absorptiometry (measurement wavelength: 450 nm, control wavelength: 620 nm). ) To measure the number of viable cells. Except for the operations described in detail below, the operation was performed according to the manual attached to the above measurement kit.
Specifically, 10 μL of a reagent containing "water-soluble tetrazolium salt (WST-1)" as a color-developing substrate was added to the cell culture well after the above-mentioned predetermined culture time had passed, and 5% CO _{2 was} added. Incubated for 45 minutes under 37 ° C. conditions. Then, with respect to the cell culture solution to which the color-developing reagent was added, the absorbance at a wavelength of 450 nm (A ₄₅₀ ) and the absorbance at a wavelength of 620 nm (A ₆₂₀ ) were measured using a spectrophotometer (microplate reader), and A ₄₅₀ was A _{620. The value A 450-620} corrected in 1 was calculated, and the cell viability of each test group was calculated. Then, the relative value of the cell viability (%) of each test group when the cell viability in the control group is 100% is expressed by the following formula: cell viability (%) = (A _{450-620 of} each test group). It was calculated by ÷ (A _{450-620 in the} control group) × 100 ;. The results are shown in FIG.

As shown in FIG. 5, the cell viability was almost the same in all the test groups cultured by adding 0 nM, 100 nM or 500 nM EGF in addition to the exosomes of Sample 1 as compared with the control group. (There was no significant difference in cell viability). From this, it was confirmed that the exosomes (that is, CD63-GFP-exosomes) and EGF related to Sample 1 added to the medium (supplied to the target cells) had no effect on cell proliferation of the target cells. That is, it was confirmed that the CD63-GFP-exosomes taken up in the medium and in the cells had no cytotoxicity and cell proliferation promoting activity. It was also confirmed that EGF added in addition to CD63-GFP-exosome did not promote cell proliferation of the target cells.

<Test 10: Intracellular introduction test of foreign substances using exosomes>
Using the exosomes of Sample 2, the efficiency of supplying foreign substances contained in the exosomes into cells was evaluated. Specifically, by using saporin as a foreign substance and examining the survival rate of the cells when the saporin-encapsulating exosomes were supplied to the cells, the efficiency of the saporins contained in the exosomes being taken up into the cells was evaluated. Here, saporin is a kind of proteinaceous toxin (that is, a kind of substance having antitumor activity), and it is known that cells that have taken up saporin cause cell death. That is, the lower the cell viability, the more efficiently the saporin was introduced into the cells.
As a test method, the cell viability (%) of the test cells was measured under the same conditions and methods as in Test 9 above, except that the culture in the presence of exosomes (main culture) was changed to the following conditions. That is, in this test, the medium in the culture vessel after preculture was prepared with 4 μg / mL exosomes (that is, saporin-encapsulating exosomes) or 7 μg / mL saporin according to Sample 2 and 0 nM (that is, without EGF addition) or 500 nM. The conditions were the same as in Test 9 above, except that the medium was replaced with A431 medium containing EGF (that is, MEM medium containing 10% FBS) and cultured for 48 hours. Here, each test group will be referred to as a test group 10-1 to a test group 10-4. As a control group, a test group containing neither exosome (exosome according to sample 2, that is, saporin-encapsulating exosome), saporin, or EGF was provided in the medium. The composition of the medium used for the main culture of each test group is summarized in Table 4.

For each test group after the main culture, the relative value of the cell viability (%) of each test group was calculated when the cell viability in the control group was set to 100% by the same method as in Test 9. The results are shown in FIG. Here, the bar graph in the figure shows the results of the control group, the test group 10-1, 10-2, 10-3, and 10-4 from the left.

As shown in FIG. 6, the cell viability of the test group 10-2 is remarkable as compared with the other test groups (control group, test group 10-1, test group 10-3, and test group 10-4). Was low (cell viability was less than half). This indicates that saporin was introduced into the cells of the test group 10-2 with particularly high efficiency as compared with the other test groups. Furthermore, from the comparison of the results of Test Group 10-1 and Test Group 10-2 and the comparison of the results of Test Group 10-3 and Test Group 10-4, by supplying EGF to the target cells, there was no EGF. It was confirmed that the uptake of saporin into cells can be promoted as compared with the case of addition. This indicates that EGF can induce macropinocytosis in target cells with high efficiency.
Here, from the results of Test Group 10-3, it was confirmed that the saporin was hardly taken up (introduced) into the cells by simply adding the saporin to the medium. On the other hand, from the comparison of the results of Test Group 10-1 and Test Group 10-3 and the comparison of the results of Test Group 10-2 and Test Group 10-4, the saporin was included in the exosome. It was confirmed that saporin can be efficiently introduced into cells.
From the above results, it was confirmed that the foreign substance can be introduced into the target cell with high efficiency by supplying the exosome containing the foreign substance and the substance that induces macropinocytosis to the target cell.

<Test 11: Intracellular uptake test of EGF-encapsulating exosomes>
Using the exosomes according to Sample 3 (that is, EGF-encapsulating exosomes), the uptake of the exosomes by the target cells was evaluated by analysis using a flow cytometer. A431 cells were used as test cells. The details of the test are as follows.

First, A431 cells pre-cultured for 1 day were prepared under the same conditions and methods as in Test 6 above, and the cells were washed 3 times using MEM medium. It also comprises 20 μg / mL CD63-GFP-exosomes (ie, sample 1), 20 μg / mL EGF-encapsulating exosomes (ie, sample 3) or 20 μg / mL transferrin (Tf) -encapsulating exosomes (ie, sample 5). 200 μL of A431 medium (MEM medium containing 10% FBS) was added per well, and the cells _{were cultured under the conditions of 5% CO 2} , 37 ° C. for 24 hours. Each test group will be referred to as a sample 1 addition group, a sample 3 addition group, and a sample 5 addition group, respectively.
The cells after culturing in the presence of the exosomes were analyzed using a flow cytometer under the same conditions and methods as in Test 6 above.
The results are shown in FIG. The graph shown in FIG. 7 shows the relative value of the average GFP fluorescence intensity per cell in the sample 3 addition group and the sample 5 addition group when the average GFP fluorescence intensity per cell in the sample 1 addition group is 100. It is a thing.

As shown in FIG. 7, the average GFP fluorescence intensity per cell in the sample 3 addition group was significantly higher than the average GFP fluorescence intensity per cell in the sample 1 addition group. Specifically, the average GFP fluorescence intensity per cell in the sample 3 addition group was about 8 times the average GFP fluorescence intensity per cell in the sample 1 addition group. This means that by supplying an exosome containing EGF to a target cell, the exosome (typically, the EGF contained in the exosome) induces macropinocytosis of the target cell, and the exosome by the cell is induced. It shows that the uptake of was promoted. That is, it is possible to suitably induce macropinocytosis in the target cell by supplying the target cell with an exosome containing a substance that induces macropinocytosis in the target cell. It was also confirmed that the induction of macropinocytosis can promote the uptake of the exosome into the target cell.
In addition, the average GFP fluorescence intensity per cell in the sample 5 addition group was slightly increased (approximately 1.9 times) as compared with the average GFP fluorescence intensity per cell in the sample 1 addition group, but the sample. It was not as remarkable as the 3 addition group. In other words, the average GFP fluorescence intensity per cell in the sample 3 addition group was higher than the average GFP fluorescence intensity per cell in sample 5 (approximately 4.3 times). Here, transferrin is a substance that induces clathrin-dependent endocytosis by stimulating the transferrin receptor. That is, from the comparison between the sample 3 addition group and the sample 5 addition group, it was confirmed that exosomes are more efficiently taken up into cells by macropinocytosis than by clathrin-dependent endocytosis.

<Test 12: Intracellular introduction test of foreign substances using EGF-encapsulating exosomes>
Efficiency of introducing foreign substances contained in the exosome into the cell using the exosome according to sample 2 (that is, the exosome containing saporin) and the exosome related to sample 4 (that is, the exosome containing both EGF and saporin). Was evaluated. Specifically, by using saporin as a foreign substance and examining the survival rate of cells when the exosome containing such saporin was supplied to the cell, the efficiency of the saporin contained in the exosome being taken up into the cell was evaluated. .. A431 cells were used as test cells. The details of the test are as follows.

As a test method, the cell viability of the test cells was measured by the same conditions and methods as in Test 10 above, except that the culture in the presence of exosomes was changed to the following conditions. Specifically, for the cells after pre-culture, the medium in the culture vessel is A431 medium containing 0.4 μg / mL, 4 μg / mL, or 20 μg / mL of sample 2 or sample 4 (that is, 10). The cell viability of the test cells was measured under the same conditions and methods as in Test 10 above, except that the cells were replaced with% FBS-containing MEM medium) and cultured for 72 hours. As a control group, a test group was provided in which the medium did not contain any of the exosomes of Sample 2 and Sample 4.

For each test group after culturing in the presence of the above exosomes, the relative value of the cell viability (%) of each test group was calculated when the cell viability in the control group was 100%. The results are shown in FIG.

In addition, the state of the cells in each of the above test groups after culturing for 72 hours in the presence of exosomes was observed using an optical microscope (phase-contrast inverted microscope). The results of microscopic observation in each test group are shown in FIG.

As shown in FIG. 8, the cells in the sample 4 addition group had lower cell viability as compared with the cells in the control group and the sample 2 addition group having the same concentration. Further, when the exosome concentration of sample 4 in the medium is in the range of at least 0.4 to 20 μg / mL, the cell viability of the target cells supplied with the exosomes decreases, and the cell viability is in the medium. It was confirmed that the concentration of exosomes related to sample 4 decreased as the concentration of exosomes related to sample 4 increased (depending on the concentration of exosomes related to sample 4). In particular, when the exosome concentration in the medium was 4 μg / mL or more (4 to 20 μg / mL), the cell viability was significantly reduced.
Further, as shown in FIG. 9, the cells in the sample 4 addition group had a non-uniform cell morphology as compared with the cells in the sample 2 addition group having the same concentration (typically, the cell size was reduced). The edges of the cells were unclear), and many cells were observed glowing white. The cells in which the morphology of such cells is heterogeneous and are observed to glow white are cells that are undergoing cell death (cell death is occurring).
These facts indicate that by using an exosome containing EGF, the saporin contained in the exosome can be efficiently introduced into a target cell. From the above results, it was confirmed that the foreign substance can be efficiently introduced into the target cell by using an exosome containing a substance that induces macropinocytosis and a foreign substance. In other words, it was confirmed that an exosome containing a substance that induces macropinocytosis and a foreign substance can be suitably used for the purpose of introducing the foreign substance into a target cell.

<Test 13: Exosome uptake test>
The culture period was extended in the presence of substances that induce exosomes and macropinocytosis, and the uptake of exosomes by the test cells under these conditions was evaluated. As the evaluation method, fluorescence observation using a confocal microscope and analysis using a flow cytometer were performed. A431 cells were used as test cells. The details of the test are as follows.

The analysis using the flow cytometer was performed as follows. The pre-cultured cells were replaced with A431 medium (ie, 10% FBS-containing MEM medium) containing 20 μg / mL exosomes of sample 1 and 100 nM or 500 nM EGF and every 24 hours (ie, exosomes). Same as Test 6 above, except that the medium was cultured with the same concentration (100 nM or 500 nM) of EGF added 24 hours, 48 hours, 72 hours, and 96 hours after the start of the culture in the presence of the medium. The cells were then cultured in the presence of exosomes for 96 hours. In addition, as a control group, a test group (hereinafter, also referred to as an EGF-free group) containing no EGF in the medium (containing exosomes according to sample 1 of 20 μg / mL) was provided.

The cells in each test group after the main culture in the presence of the above exosomes were evaluated for the uptake of exosomes by the cells by analysis using a flow cytometer in the same manner as in the above test 6. The results are shown in FIG. The graph shown in FIG. 10 shows the relative value of the average GFP fluorescence intensity per cell in each test group when the average GFP fluorescence intensity per cell in the EGF-free group is set to 100.

In addition, the cells in each of the above test groups were evaluated for the uptake of exosomes by the test cells by fluorescence observation using a confocal microscope by the same method as in Test 5. FIG. 11 shows the results of fluorescence observation of the test group to which 500 nM EGF was added and the group to which EGF was not added. These images are micrographs (images) of the intracellular uptake of exosomes according to Sample 1 in each test group. The lower row shows the results of the test group to which 500 nM EGF was added, and the upper row shows the results of the EGF-free group. Specifically, the photograph (image) shown in the left column is a micrograph in which the localization of exosomes according to Sample 1 is investigated. The photographs (images) shown in the middle column are images of nuclear staining by Hoechst 33342. In the photograph (image) shown in the right column, the image of examining the localization of exosomes related to sample 1 shown in the left column and the image of nuclear staining by Hoechst33342 shown in the center column were superimposed (merged). It is an image.

As shown in FIGS. 10 and 11, the target cells cultured for a long period of time in the presence of the exosomes and EGF according to Sample 1 had a significantly higher amount of exosome uptake as compared with the cells in the EGF-free group. It was confirmed (about 40 times or more). In particular, comparing the results of Test 6 above with the results of analysis using a flow cytometer in this test, it is possible to obtain more results from the target cells by culturing for a long period of time under the condition that the exosomes and EGF according to Sample 1 are present. It was confirmed that many exosomes can be taken up (introduced). Based on the above results, the foreign substance can be efficiently supplied by adjusting the period (supply interval) for supplying the exosome for introducing the foreign substance (that is, the composition containing the exosome for introducing the foreign substance) disclosed here to the target cells. It was confirmed that it could be introduced into the target cells well.

<Test 14: Exosome uptake test using MIA PaCa-2 cells as test cells>
When MIA PaCa-2 cells were used as the test cells, the uptake of exosomes by the test cells was evaluated. As the evaluation method, fluorescence observation using a confocal microscope and analysis using a flow cytometer were performed. As the exosome, the exosome according to Sample 1 was used. The details of the test are as follows.

Using MIA PaCa-2 cells as the test cells, the flow cytometer was used to obtain the uptake of exosomes by the test cells under the same conditions and methods as in Test 6 above, except that the culture conditions in the presence of exosomes were changed to the following. It was evaluated by the analysis used. Specifically, the pre-cultured MIA PaCa-2 cells were cultured for 24 hours in a medium containing 20 μg / mL of sample 1 exosomes and 0 nM (ie, EGF-free) or 500 nM EGF. , Except for the fact that a medium having the same composition as the medium used for the subculture of MIA PaCa-2 cells (hereinafter, also referred to as “MIA PaCa-2 medium”) was used for the preculture and the culture in the presence of exosomes. Evaluation was performed by the same method as in Test 6.

The results are shown in FIG. The graph shown in FIG. 12 shows the relative value of the average GFP fluorescence intensity per cell in the EGF-added group when the average GFP fluorescence intensity per cell in the EGF-free group (control group) is 100. ..

In addition, the uptake of exosomes by the cells (MIA PaCa-2 cells) related to the EGF-added group was evaluated by fluorescence observation using a confocal microscope. The cells used for such fluorescence observation were dextran (Texas red Dextran) labeled with a fluorescent dye (Texas Red) at a concentration of 0.5 mg / mL in addition to the exosome according to Sample 1 at 20 μg / mL and EGF at 500 nM. ) Was cultured in MIA PaCa-2 medium for 24 hours. Fluorescence observation using a confocal microscope was carried out by the same method as in Test 5 above. The results are shown in FIG. These images are micrographs (images) of the intracellular uptake of exosomes and dextran according to Sample 1 in this test. Specifically, the photograph (image) shown on the far left is a micrograph in which the localization of exosomes related to Sample 1 is investigated. The second photograph (image) from the left is an image showing the localization of Texas red-dextran. The second photograph (image) from the right is an image of nuclear staining by Hoechst 33342. The photograph (image) shown on the far right is an image of examining the localization of exosomes shown on the far left, an image of examining the localization of Texas red-dextran shown second from the left, and the above right. It is an image superimposed (merged) with the nuclear staining by Hoechst 33342 shown in the second.

As shown in FIGS. 12 and 13, it was confirmed that EGF promoted the uptake of exosomes even when MIA PaCa-2 cells were used as test cells (that is, target cells). This indicates that macropinocytosis can be induced by EGF even when MIA PaCa-2 cells are used as the target cells.

<Test 15: Exosome uptake test using BxPC-3 cells as test cells>
When BxPC-3 cells were used as the test cells, the uptake of exosomes by the test cells was evaluated. As an evaluation method, an analysis using a flow cytometer was performed. As the exosome, the exosome according to Sample 1 was used. The details of the test are as follows.

BxPC-3 cells were used as the test cells, and the uptake of exosomes by the test cells was performed using a flow cytometer under the same conditions and methods as in Test 6 above, except that the culture conditions in the presence of exosomes were changed to the following. It was evaluated by the analysis. Specifically, the pre-cultured BxPC-3 cells were cultured for 24 hours in a medium containing 20 μg / mL of Exosome according to Sample 1 and 0 nM (that is, EGF-free) or 500 nM EGF. The above test 6 except that a medium having the same composition as the medium used for the subculture of BxPC-3 cells (hereinafter, also referred to as “BxPC-3 cell medium”) was used for the preculture and the culture in the presence of exosomes. It was evaluated by the same method as.

The results are shown in FIG. The graph shown in FIG. 14 shows the relative value of the average GFP fluorescence intensity per cell in the EGF-added group when the average GFP fluorescence intensity per cell in the EGF-free group (control group) is 100. ..

As shown in FIG. 14, it was confirmed that EGF promoted the uptake of exosomes even when BxPC-3 cells were used as test cells (that is, target cells). This indicates that macropinocytosis can be induced by EGF even when BxPC-3 cells are used as the target cells.

Here, from the results of Test 14 and Test 15, the result of the test using A431 cells as the test cells is not a phenomenon peculiar to the A431 cells, but other cells (typically human-derived cells, general). It was confirmed that it is also applicable to tumor cells, particularly MIA PaCa-2 cells and BxPC-3 cells). That is, the results of Test 14 and Test 15 show that the target cells in the present invention are not limited to A431 cells, but other cells (typically human-derived cells, generally tumor cells, especially MIA PaCa-2 cells). And in BxPC-3 cells), by supplying a substance that induces macropinocytosis to the test cell and inducing macropinocytosis in the cell, a foreign substance contained in the exosome is introduced into the cell. It shows that it can be introduced suitably. Further, even when cells other than A431 cells (typically human-derived cells, generally tumor cells, particularly MIA PaCa-2 cells and BxPC-3 cells) are used as the target cells in the present invention. It is shown that by supplying an exosome containing a substance that induces macropinocytosis and a foreign substance to a target cell, the foreign substance contained in the exosome can be efficiently introduced into the target cell.

<Test 16: Macropinocytosis induction test in SDF>
The uptake of exosomes by test cells when SDF was used as a substance that induces macropinocytosis was evaluated by analysis using a flow cytometer. As the exosome, the exosome related to Sinepull 1 was used. In addition, HeLa cells were used as test cells. The details of the test are as follows.
First, HeLa cells were seeded in a 24-well culture dish (24-well plate) at ^{a cell density of 4.7 × 10 4} cells / well, and cultured for 1 day in an incubator under the conditions of _{5% CO 2 and 37 ° C.} As the medium, 1.0 mL of HeLa medium was used per well.
For HeLa cells after the above 1-day culture (preculture), the medium in the culture vessel was removed, and the inside of the culture vessel was washed 3 times with α-MEM medium (200 μL). Then, 200 μL of a medium containing 20 μg / mL of sample 1 exosome and 100 nM or 200 nM SDF was added per well, and the cells were _{cultured for 24 hours under the conditions of 5% CO 2} and 37 ° C. (main culture). .. Here, as the medium, a HeLa medium (that is, containing 10% FBS) was used. Further, as a control group, a test group (hereinafter, also referred to as “SDF-free group”) containing no SDF in the medium (containing exosomes according to sample 1 of 20 μg / mL) was provided.

For HeLa cells after culturing in the presence of the above exosomes, the uptake of exosomes in the target cells was analyzed using a flow cytometer in the same manner as in Test 6 above. The results are shown in FIG. The graph shown in FIG. 15 shows the relative value of the average GFP fluorescence intensity per cell in each test group when the average GFP fluorescence intensity per cell in the SDF-free group was set to 100.

As shown in FIG. 15, the average GFP fluorescence intensity per cell in the SDF-added group was higher than the average GFP fluorescence intensity per cell in the SDF-free group. It was also confirmed that the average GFP fluorescence intensity per cell in the SDF-added group increased (depending on the SDF concentration in the medium) as the concentration of SDF in the medium increased. This indicates that macropinocytosis can be induced by supplying SDF to the target cells. That is, it is shown that SDF can be a substance that induces macropinocytosis, similar to EGF described above. From the above results, it was confirmed that SDF can be suitably used as a substance that induces macropinocytosis.

As described above, according to the techniques disclosed herein, the uptake of exosomes by macropinocytosis is utilized to efficiently introduce a foreign substance (typically a substance having pharmacological activity) into a target cell. be able to. Further, according to the technique disclosed here, a composition for efficiently introducing a foreign substance (typically a substance having pharmacological activity) into a target cell by utilizing the uptake of exosomes by macropinocytosis. It is possible to provide an exosome capable of efficiently introducing a substance and a foreign substance into a target cell.
Therefore, the techniques disclosed herein can be suitably used in a drug delivery system for introducing a foreign substance (typically a substance having pharmacological activity) into a target cell. For example, by using a substance having an anticancer (antitumor) action, a substance having a differentiation inducing action, or the like as a foreign substance, it can be suitably used in the fields of cancer treatment and regenerative medicine.

Claims (7)

An exosome used to introduce a foreign substance into a target cell from outside the cell into the cell.
With one or more foreign substances
Substances that induce macropinocytosis in target cells and
Exosomes that contain all of them. The exosome according to claim 1, wherein the substance that induces macropinocytosis in the target cell is a substance that stimulates the epidermal growth factor (EGF) receptor or a substance that stimulates the CXC chemokine receptor 4. The exosome according to claim 1 or 2, wherein the substance that induces macropinocytosis in the target cell is EGF or an analog thereof, or SDF or an analog thereof. The exosome according to any one of claims 1 to 3, wherein the foreign substance is a substance having pharmacological activity. The exosome according to any one of claims 1 to 4, wherein the target cell is a human tumor cell and the foreign substance is a compound having antitumor activity. A composition used for introducing a foreign substance into a target cell from the outside of the cell into the cell.
With one or more pharmaceutically acceptable carriers
Exosomes containing both substances that induce macropinocytosis in target cells and one or more foreign substances, and
A composition comprising. A method of introducing a foreign substance into a target cell in vitro.
Prepare a cell culture containing the target cells, and include in the cell culture a substance that induces macropinocytosis with respect to the target cells and one or more foreign substances. Supplying exosomes,
Including methods.

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